Vaginal inserted estradiol pharmaceutical compositions and methods

ABSTRACT

Disclosed herein is, among other things, a soft gel vaginal pharmaceutical composition and dosage form containing solubilized estradiol for the treatment of vulvovaginal atrophy (VVA) and female sexual dysfunction (FSD).

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/372,385, filed Dec. 7, 2016, which is a continuation-in-partof U.S. Pat. Appl. No. 14/521,230, filed Oct. 22, 2014; and claimspriority to U.S. Provisional Pat. Appl. No. 62/264,309, filed Dec. 7,2015; U.S. Provisional Pat. Appl. No. 62/296,552, filed Feb. 17, 2016;U.S. Provisional Pat. Appl. No. 62/324,838, filed Apr. 19, 2016; U.S.Provisional Pat. Appl. No. 62/329,940, filed Apr. 29, 2016; and U.S.Provisional Pat. Appl. No. 62/348,820, filed Jun. 10, 2016; whichapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This application is directed to pharmaceutical compositions, methods,and devices related to hormone replacement therapy.

BACKGROUND OF THE INVENTION

Postmenopausal women frequently suffer from atrophic vaginitis or vulvarand vaginal atrophy (hereinafter “vulvovaginal atrophy” or “VVA”) withsymptoms including, for example, vaginal dryness, vaginal odor, vaginalor vulvar irritation or itching, dysuria (pain, burning, or stingingwhen urinating), dyspareunia (vaginal pain associated with sexualactivity), or vaginal bleeding associated with sexual activity. Othersymptoms include soreness; with urinary frequency and urgency; urinarydiscomfort and incontinence also occurring (“estrogen-deficient urinarystate(s)”). One symptom of vaginal atrophy is an increased vaginal pH,which creates an environment more susceptible to infections. The mucosalepithelium of the VVA patients also reported to show signs of severeatrophy and upon cytological examination accompanied by an increasednumber of the parabasal cells and a reduced number of superficial cells.

Each of these VVA-related states manifest symptoms associated withdecreased estrogenization of the vulvovaginal tissue, and can even occurin women treated with oral administration of an estrogen-basedpharmaceutical drug product. Although VVA is most common with menopausalwomen, it can occur at any time in a woman's life cycle. VVA symptomsalso interfere with sexual activity and satisfaction. Women with femalesexual dysfunction (FSD) are almost 4 times more likely to have VVA thanthose without FSD.

Estrogen treatment has proven to be very successful in controllingmenopausal symptoms, including VVA and FSD. Several studies have shownthat the symptoms connected with vaginal atrophy are often relieved byestrogen treatment given either systemically or topically. The existingtreatments have numerous problems, for example compliance issues withpatients not completing or continuing treatment due to the problemsassociated with the form of treatment.

Accordingly, there remains a need in the art for treatments for VVA andFSD that overcome these limitations.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is, among other things, a new soft gel vaginalpharmaceutical composition and dosage form containing solubilizedestradiol for the treatment of VVA. The soft gel vaginal pharmaceuticalcomposition has been designed to mitigate common limitations found withother vaginal forms of estradiol. The soft gel vaginal pharmaceuticalcomposition eases vaginal administration, provides improved safety ofinsertion, minimizes vaginal discharge following administration, andprovides a more effective dosage form having improved efficacy, safetyand patient compliance.

According to various aspects and embodiments of this disclosure, a softgel vaginal pharmaceutical composition as a treatment forpost-menopausal women suffering with moderate to severe symptoms of VVAis provided.

Provided herein is a suppository comprising: a) a therapeuticallyeffective amount of estradiol; and b) a solubilizing agent comprising amedium chain oil.

In some embodiments, the suppository includes about 1 μg to about 25 μgof estradiol. For example, the suppository can include about 1 μg toabout 10 μg of estradiol; and about 10 μg to about 25 μg of estradiol.

In some embodiments, the estradiol is solubilized.

In some embodiments, the medium chain oil includes at least one C6-C12fatty acid or a glycol, monoglyceride, diglyceride, or triglycerideester thereof.

In some embodiments, the solubilizing agent includes at least one esterselected from the group consisting of: an ester of caproic fatty acid,an ester of caprylic fatty acid, an ester of capric fatty acid, andcombinations thereof. For example, the solubilizing agent can include acaprylic/capric triglyceride.

In some embodiments, the suppository further includes a capsule. Forexample, the capsule can be a soft gelatin capsule.

Also provided herein is a suppository comprising: a) a therapeuticallyeffective amount of estradiol; b) a caprylic/capric triglyceride; c) anon-ionic surfactant comprising PEG-6 palmitostearate and ethyleneglycol palmitostearate; and d) a soft gelatin capsule.

In some embodiments, a suppository provided herein includes about 25 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estradiol of about 19pg*hr/mL to about 29 pg*hr/mL; and 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estradiol of about 75 pg*hr/mL to about 112pg*hr/mL.

In some embodiments, a suppository provided herein includes about 25 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone of about 9 pg*hr/mLto about 14 pg*hr/mL; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estrone of about 43 pg*hr/mL to about 65 pg*hr/mL.

In some embodiments, a suppository provided herein includes about 25 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone sulfate of about 416pg*hr/mL to about 613 pg*hr/mL; and 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estrone sulfate of about 3598 pg*hr/mL toabout 5291 pg*hr/mL.

In some embodiments, a suppository provided herein includes about 10 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estradiol of about 12pg*hr/mL to about 18 pg*hr/mL; and 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estradiol of about 42 pg*hr/mL to about 63pg*hr/mL. In some embodiments, the suppository further provides acorrected geometric mean time to peak plasma concentration (T_(max)) ofestradiol of about 1 hrs to about 3 hrs.

In some embodiments, a suppository provided herein includes about 10 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone of about 4 pg*hr/mLto about 7 pg*hr/mL; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estrone of about 20 pg*hr/mL to about 31 pg*hr/mL. Insome embodiments, the suppository further provides a corrected geometricmean time to peak plasma concentration (T_(max)) of estrone of about 4hrs to about 8 hrs.

In some embodiments, a suppository provided herein includes about 10 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone sulfate of about 10pg*hr/mL to about 16 pg*hr/mL; and 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estrone sulfate of about 56 pg*hr/mL toabout 84 pg*hr/mL. In some embodiments, the suppository further providesa corrected geometric mean time to peak plasma concentration (T_(max))of estrone sulfate of about 4 hrs to about 7 hrs.

In some embodiments, a suppository provided herein includes about 4 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estradiol of about 4pg*hr/mL to about 8 pg*hr/mL; and 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estradiol of about 16 pg*hr/mL to about 26pg*hr/mL. In some embodiments, the suppository further provides acorrected geometric mean time to peak plasma concentration (T_(max)) ofestradiol of about 0.25 hrs to about 2 hrs.

In some embodiments, a suppository provided herein includes about 4 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone of about 1 pg*hr/mLto about 3 pg*hr/mL; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estrone of about 8 pg*hr/mL to about 13 pg*hr/mL. Insome embodiments, the suppository further provides a corrected geometricmean time to peak plasma concentration (T_(max)) of estrone of about 1hrs to about 4 hrs.

In some embodiments, a suppository provided herein includes about 4 μgof estradiol, wherein administration of the suppository to a patientprovides, in a plasma sample from the patient: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone sulfate of about 4pg*hr/mL to about 7 pg*hr/mL; and 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estrone sulfate of about 22 pg*hr/mL toabout 34 pg*hr/mL. In some embodiments, the suppository further providesa corrected geometric mean time to peak plasma concentration (T_(max))of estrone sulfate of about 1 hrs to about 3 hrs.

Also provided herein is a suppository comprising about 1 μg to about 25μg of estradiol, wherein administration of the suppository to a patientprovides a corrected geometric mean peak plasma concentration (C_(max))of estradiol that is less than about 30 pg*hr/mL. For example,administration of the suppository to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estradiol that isless than about 18 pg*hr/mL.

In some embodiments, a suppository comprising about 1 μg to about 25 μgof estradiol is provided, wherein administration of the suppository to apatient provides a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estradiol that is less than about 112 pg*hr/mL. Forexample, administration of the suppository to a patient provides acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiolthat is less than about 63 pg*hr/mL.

In some embodiments, a suppository comprising about 1 μg to about 25 μgof estradiol is provided, wherein administration of the suppository to apatient provides a corrected geometric mean peak plasma concentration(C_(max)) of estrone that is less than about 14 pg*hr/mL. For example,administration of the suppository to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estrone that isless than about 7 pg*hr/mL.

In some embodiments, a suppository comprising about 1 μg to about 25 μgof estradiol is provided, wherein administration of the suppository to apatient provides a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone that is less than about 65 pg*hr/mL. For example,administration of the suppository to a patient provides a correctedgeometric mean area under the curve (AUC)₀₋₂₄ of estrone that is lessthan about 31 pg*hr/mL.

In some embodiments, a suppository comprising about 1 μg to about 25 μgof estradiol is provided, wherein administration of the suppository to apatient provides a corrected geometric mean peak plasma concentration(C_(max)) of estrone sulfate that is less than about 613 pg*hr/mL. Forexample, administration of the suppository to a patient provides acorrected geometric mean peak plasma concentration (C_(max)) of estronesulfate that is less than about 16 pg*hr/mL.

In some embodiments, a suppository comprising about 1 μg to about 25 μgof estradiol is provided, wherein administration of the suppository to apatient provides a corrected geometric mean area under the curve(AUC)0-24 of estrone sulfate that is less than about 5291 pg*hr/mL. Forexample, administration of the suppository to a patient provides acorrected geometric mean area under the curve (AUC)0-24 of estronesulfate that is less than about 84 pg*hr/mL.

Further provided herein is a suppository comprising about 1 μg to about25 μg of estradiol, wherein administration of the suppository to theproximal region of the vagina of a patient provides a therapeuticallyeffective concentration of estradiol over 24 hours in the proximalregion of the vagina.

This disclosure also provides a method of treating an estrogen-deficientstate, the method comprising administering to a patient in need thereof,a suppository as provided herein. In some embodiments, a method oftreating vulvovaginal atrophy is provided, the method comprisingadministering to a patient in need thereof, a suppository as providedherein.

In some embodiments of the methods provided herein, treatment includesreducing the severity of one or more symptoms selected from the groupconsisting of: vaginal dryness, dyspareunia, vaginal or vulvarirritation, vaginal or vulvar burning, vaginal or vulvar itching,dysuria, and vaginal bleeding associated with sexual activity.

In some embodiments of the methods provided herein treatment includesreducing the vaginal pH of the patient. For example, treatment includesreducing the vaginal pH of the patient to a pH of less than about 5.0.

In some embodiments of the methods provided herein treatment includes achange in cell composition of the patient. For example, the change incell composition includes reducing the number of parabasal vaginal cellsor increasing the number of superficial vaginal cells. In someembodiments, the number of parabasal vaginal cells in the patient arereduced by at least about 35% (e.g., at least about 50%). In someembodiments, the number of superficial vaginal cells are increased by atleast about 5% (e.g., at least about 35%).

Further provided herein is a method for reducing vaginal dischargefollowing administration of a suppository, the method comprisingadministering to a patient in need thereof, a suppository providedherein, wherein the vaginal discharge following administration of thesuppository is compared to the vaginal discharge followingadministration of a reference drug.

Also provided herein is a method for treating female sexual dysfunctionin a female subject in need thereof. The method includes administeringto the subject a vaginal suppository as described herein. In someembodiments, the method includes administering to the subject a vaginalsuppository comprising: (a) a pharmaceutical composition comprising: atherapeutically effective amount of estradiol; a caprylic/caprictriglyceride; a non-ionic surfactant comprising PEG-6 palmitostearateand ethylene glycol palmitostearate; and (b) a soft gelatin capsule;wherein the vaginal suppository includes from about 1 microgram to about25 micrograms of estradiol; wherein estradiol is the only active hormonein the vaginal suppository. In some embodiments, the vaginal suppositorydoes not include a hydrophilic gel-forming bioadhesive agent in thesolubilizing agent. In some embodiments, treating female sexualdysfunction includes increasing the subject's desire, arousal,lubrication, satisfaction, and or/orgasms.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned features and objects of the this disclosure willbecome more apparent with reference to the following description takenin conjunction with the accompanying drawings wherein like referencenumerals denote like elements and in which:

FIG. 1 is a flow diagram illustrating a process in accordance withvarious embodiments of the invention;

FIG. 2 illustrates a suppository in accordance with various embodimentsof the invention;

FIG. 3 is a linear plot of mean plasma estradiol—baseline adjustedconcentrations versus time (N=34);

FIG. 4 is a semi-logarithmic plot of mean plasma estradiol—baselineadjusted concentrations versus time (N=34);

FIG. 5 is a linear plot of mean plasma estrone—baseline adjustedconcentrations versus time (N=33);

FIG. 6 is a semi-logarithmic plot of mean plasma estrone—baselineadjusted concentrations versus time (N=33);

FIG. 7 is a linear plot of mean plasma estrone sulfate—baseline adjustedconcentrations versus time (N=24); and

FIG. 8 is a semi-logarithmic plot of mean plasma estronesulfate—baseline adjusted concentrations versus time (N=24).

FIG. 9 is a study schematic diagram.

FIG. 10 shows the percentage change in superficial cells at 12 weekscompared to placebo.

FIG. 11 shows the percentage change in superficial cells at week 2, week6, week 8, and week 12 compared to placebo.

FIG. 12 shows percentage change in superficial cells per dose for eachof week 2, week 6, week 8, and week 12 compared to placebo.

FIG. 13 shows the percentage change in parabasal cells at 12 weekscompared to placebo.

FIG. 14 shows the percentage change in parabasal cells at week 2, week6, week 8, and week 12 compared to placebo.

FIG. 15 shows the percentage change in parabasal cells per dose for eachof week 2, week 6, week 8, and week 12 compared to placebo

FIG. 16 shows the percentage change in pH at 12 weeks compared toplacebo.

FIG. 17 shows the percentage change in pH at week 2, week 6, week 8, andweek 12 compared to placebo.

FIG. 18 shows the percentage change in pH per dose for each of week 2,week 6, week 8, and week 12 compared to placebo.

FIG. 19A shows the change in visual assessments from baseline to week 12in vaginal color in a modified item to treat (MITT) population.

FIG. 19B shows the change in visual assessments from baseline to week 12in vaginal epithelial integrity in a modified item to treat (MITT)population.

FIG. 19C shows the change in visual assessments from baseline to week 12in vaginal epithelial thickness a modified item to treat (MITT)population.

FIG. 19D shows the change in visual assessments from baseline to week 12in vaginal secretions in a modified item to treat (MITT) population.

FIG. 20A shows the correlation between the total sum of four visualassessments and dyspareunia at week 12 in an intent to treat (ITT)population.

FIG. 20B shows the correlation between the total sum of four visualassessments and vaginal dryness at week 12 in an intent to treat (ITT)population.

FIG. 21 shows baseline adjusted estradiol serum concentration (pg/mL)assessed on Day 1 (squares) and Week 12 (diamonds) for four treatmentarms.

FIG. 22 shows baseline adjusted estradiol serum concentration (pg/mL)assessed on Day 14 (squares) and Week 12 (diamonds) for four treatmentarms.

FIG. 23 shows estradiol plasma levels measured in subjects following asupine period after administration of the estradiol formulation,compared with plasma levels measured in subjects following an ambulatoryperiod after administration of the estradiol formulation.

FIG. 24 shows mean change from baseline in Total FSFI score at Week 12.

FIG. 25A shows the mean change from baseline to week 12 in theindividual FSFI lubrication score.

FIG. 25B shows the mean change from baseline to week 12 in theindividual FSFI arousal score.

FIG. 25C shows the mean change from baseline to week 12 in theindividual FSFI satisfaction score.

FIG. 25D shows the mean change from baseline to week 12 in theindividual FSFI desire score.

FIG. 25E shows the mean change from baseline to week 12 in theindividual FSFI orgasm score.

FIG. 26A shows an estradiol softgel capsule held with the larger endbetween the fingers.

FIG. 26B shows insertion of an estradiol softgel capsule in a recliningposition. The softgel is inserted into the lower third of the vaginawith the smaller end up.

FIG. 26C shows insertion of an estradiol softgel capsule in a standingposition. The softgel is inserted into the lower third of the vaginawith the smaller end up.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description of embodiments of this disclosure,reference is made to the accompanying drawings in which like referencesindicate similar elements, and in which is shown by way of illustrationspecific embodiments in which this disclosure may be practiced. Theseembodiments are described in sufficient detail to enable those skilledin the art to practice this disclosure, and it is to be understood thatother embodiments may be utilized and that other changes may be madewithout departing from the scope of the this disclosure. The followingdetailed description is, therefore, not to be taken in a limiting sense,and the scope of this disclosure is defined only by the appended claims.As used in this disclosure, the term “or” shall be understood to bedefined as a logical disjunction (i.e., and/or) and shall not indicatean exclusive disjunction unless expressly indicated as such with theterms “either,” “unless,” “alternatively,” and words of similar effect.

I. DEFINITIONS

The term “active pharmaceutical ingredient” (“API”) as used herein,means the active compound(s) used in formulating a drug product.

The term “co-administered” as used herein, means that two or more drugproducts are administered simultaneously or sequentially on the same ordifferent days.

The term “drug product” as used herein means at least one activepharmaceutical ingredient in combination with at least one excipient andprovided in unit dosage form.

The term “area under the curve” (“AUC”) refers to the area under thecurve defined by changes in the blood concentration of an activepharmaceutical ingredient (e.g., estradiol or progesterone), or ametabolite of the active pharmaceutical ingredient, over time followingthe administration of a dose of the active pharmaceutical ingredient.“AUC_(1-∞)” is the area under the concentration-time curve extrapolatedto infinity following the administration of a dose. “AUC_(0-t)” is thearea under the concentration-time curve from time zero to time tfollowing the administration of a dose, wherein t is the last time pointwith a measurable concentration.

The term “C_(max)” refers to the maximum value of blood concentrationshown on the curve that represents changes in blood concentrations of anactive pharmaceutical ingredient (e.g., progesterone or estradiol), or ametabolite of the active pharmaceutical ingredient, over time.

The term “T_(max)” refers to the time that it takes for the bloodconcentration an active pharmaceutical ingredient (e.g., estradiol orprogesterone), or a metabolite of the active pharmaceutical ingredient,to reach the maximum value.

The term “bioavailability,” which has the meaning defined in 21 C.F.R. §320.1(a), refers to the rate and extent to which an API or activeingredient or active moiety is absorbed from a drug product and becomesavailable at the site of action. For example, bioavailability can bemeasured as the amount of API in the blood (serum or plasma) as afunction of time. Pharmacokinetic (PK) parameters such as AUC, C_(max),or T_(max) may be used to measure and assess bioavailability. For drugproducts that are not intended to be absorbed into the bloodstream,bioavailability may be assessed by measurements intended to reflect therate and extent to which the API or active ingredient or active moietybecomes available at the site of action.

The term “bioequivalent,” which has the meaning defined in 21 C.F.R. §320.1(e), refers to the absence of a significant difference in the rateand extent to which the API or active ingredient or active moiety inpharmaceutical equivalents or pharmaceutical alternatives becomesavailable at the site of drug action when administered at the same molardose under similar conditions in an appropriately designed study. Wherethere is an intentional difference in rate (e.g., in certain extendedrelease dosage forms), certain pharmaceutical equivalents oralternatives may be considered bioequivalent if there is no significantdifference in the extent to which the active ingredient or moiety fromeach product becomes available at the site of drug action. This appliesonly if the difference in the rate at which the active ingredient ormoiety becomes available at the site of drug action is intentional andis reflected in the proposed labeling, is not essential to theattainment of effective body drug concentrations on chronic use, and isconsidered medically insignificant for the drug. In practice, twoproducts are considered bioequivalent if the 90% confidence interval ofthe AUC, C_(max), or optionally T_(max) is within 80.00% to 125.00%.

The term “bio-identical,” “body-identical,” or “natural” used inconjunction with the hormones disclosed herein, means hormones thatmatch the chemical structure and effect of those that occur naturally orendogenously in the human body. An exemplary natural estrogen isestradiol.

The term “bio-identical hormone” or “body-identical hormone” refers toan active pharmaceutical ingredient that is structurally identical to ahormone naturally or endogenously found in the human body (e.g.,estradiol and progesterone).

The term “estradiol” refers to (17β)-estra-1,3,5(10)-triene-3,17-diol.Estradiol is also interchangeably called 17β-estradiol, oestradiol, orE2, and is found endogenously in the human body. As used herein,estradiol refers to the bio-identical or body-identical form ofestradiol found in the human body having the structure:

Estradiol is supplied in an anhydrous or hemi-hydrate form. For thepurposes of this disclosure, the anhydrous form or the hemihydrate formcan be substituted for the other by accounting for the water or lack ofwater according to well-known and understood techniques.

The term “solubilized estradiol” means that the estradiol or a portionthereof is solubilized or dissolved in the solubilizing agent(s) or theformulations disclosed herein.

Solubilized estradiol may include estradiol that is about 80%solubilized, about 85% solubilized, about 90% solubilized, about 95%solubilized, about 96% solubilized, about 97% solubilized, about 98%solubilized, about 99% solubilized or about 100% solubilized. In someembodiments, the estradiol is “fully solubilized” with all orsubstantially all of the estradiol being solubilized or dissolved in thesolubilizing agent. Fully solubilized estradiol may include estradiolthat is about 97% solubilized, about 98% solubilized, about 99%solubilized or about 100% solubilized. Solubility can be expressed as amass fraction (% w/w, which is also referred to as wt %).

The term “progesterone” refers to pregn-4-ene-3,20-dione. Progesteroneis also interchangeably called P4 and is found endogenously in the humanbody. As used herein, progesterone refers to the bio-identical orbody-identical form of progesterone found in the human body having thestructure:

The term “solubilized progesterone” means that the progesterone or aportion thereof is solubilized or dissolved in the solubilizing agent(s)or the formulations disclosed herein. In some embodiments, theprogesterone is “partially solubilized” with a portion of theprogesterone being solubilized or dissolved in the solubilizing agentand a portion of the progesterone being suspended in the solubilizingagent. Partially solubilized progesterone may include progesterone thatis about 1% solubilized, about 5% solubilized, about 10% solubilized,about 15% solubilized, about 20% solubilized, about 30% solubilized,about 40% solubilized, about 50% solubilized, about 60% solubilized,about 70% solubilized, about 80% solubilized, about 85% solubilized,about 90% solubilized or about 95% solubilized. In other embodiments,the progesterone is “fully solubilized” with all or substantially all ofthe progesterone being solubilized or dissolved in the solubilizingagent. Fully solubilized progesterone may include progesterone that isabout 97% solubilized, about 98% solubilized, about 99% solubilized orabout 100% solubilized. Solubility can be expressed as a mass fraction(% w/w, which is also referred to as wt %).

The terms “micronized progesterone” and “micronized estradiol,” as usedherein, include micronized progesterone and micronized estradiol havingan X50 particle size value below about 15 microns or having an X90particle size value below about 25 microns. The term “X50” means thatone-half of the particles in a sample are smaller in diameter than agiven number. For example, micronized progesterone having an X50 of 5microns means that, for a given sample of micronized progesterone,one-half of the particles have a diameter of less than 5 microns.Similarly, the term “X90” means that ninety percent (90%) of theparticles in a sample are smaller in diameter than a given number.

The term “glyceride” is an ester of glycerol (1,2,3-propanetriol) withacyl radicals of fatty acids and is also known as an acylglycerol. Ifonly one position of the glycerol molecule is esterified with a fattyacid, a “monoglyceride” or “monoacylglycerol” is produced; if twopositions are esterified, a “diglyceride” or “diacylglycerol” isproduced; and if all three positions of the glycerol are esterified withfatty acids, a “triglyceride” or “triacylglycerol” is produced. Aglyceride is “simple” if all esterified positions contain the same fattyacid; whereas a glyceride is “mixed” if the esterified positionscontained different fatty acids. The carbons of the glycerol backboneare designated sn-1, sn-2 and sn-3, with sn-2 being in the middle carbonand sn-1 and sn-3 being the end carbons of the glycerol backbone.

The term “solubilizing agent” refers to an agent or combination ofagents that solubilize an active pharmaceutical ingredient (e.g.,estradiol or progesterone). For example and without limitation, suitablesolubilizing agents include medium chain oils and other solvents andco-solvents that solubilize or dissolve an active pharmaceuticalingredient to a desirable extent. Solubilizing agents suitable for usein the formulations disclosed herein are pharmaceutical gradesolubilizing agents (e.g., pharmaceutical grade medium chain oils). Itwill be understood by those of skill in the art that other excipients orcomponents can be added to or mixed with the solubilizing agent toenhance the properties or performance of the solubilizing agent orresulting formulation. Examples of such excipients include, but are notlimited to, surfactants, emulsifiers, thickeners, colorants, flavoringagents, etc. In some embodiments, the solubilizing agent is a mediumchain oil and, in some other embodiments, the medium chain oil iscombined with a co-solvent(s) or other excipient(s).

The term “medium chain” is used to describe the aliphatic chain lengthof fatty acid containing molecules. “Medium chain” specifically refersto fatty acids, fatty acid esters, or fatty acid derivatives thatcontain fatty acid aliphatic tails or carbon chains that contain 6 (C6)to 14 (C14) carbon atoms, 8 (C8) to 12 (C12) carbon atoms, or 8 (C8) to10 (C10) carbon atoms.

The terms “medium chain fatty acid” and “medium chain fatty acidderivative” are used to describe fatty acids or fatty acid derivativeswith aliphatic tails (i.e., carbon chains) having 6 to 14 carbon atoms.Fatty acids consist of an unbranched or branched aliphatic tail attachedto a carboxylic acid functional group. Fatty acid derivatives include,for example, fatty acid esters and fatty acid containing molecules,including, without limitation, mono-, di- and triglycerides that includecomponents derived from fatty acids. Fatty acid derivatives also includefatty acid esters of ethylene or propylene glycol. The aliphatic tailscan be saturated or unsaturated (i.e., having one or more double bondsbetween carbon atoms). In some embodiments, the aliphatic tails aresaturated (i.e., no double bonds between carbon atoms). Medium chainfatty acids or medium chain fatty acid derivatives include those withaliphatic tails having 6-14 carbons, including those that are C6-C14,C6-C12, C8-C14, C8-C12, C6-C10, C8-C10, or others. Examples of mediumchain fatty acids include, without limitation, caproic acid, caprylicacid, capric acid, lauric acid, myristic acid, and derivatives thereof.

The term “oil,” as used herein, refers to any pharmaceuticallyacceptable oil, especially medium chain oils, and specifically excludingpeanut oil, that can suspend or solubilize bioidentical progesterone orestradiol, including starting materials or precursors thereof, includingmicronized progesterone or micronized estradiol as described herein.

The term “medium chain oil” refers to an oil wherein the composition ofthe fatty acid fraction of the oil is substantially medium chain (i.e.,C6 to C14) fatty acids, i.e., the composition profile of fatty acids inthe oil is substantially medium chain. As used herein, “substantially”means that between 20% and 100% (inclusive of the upper and lowerlimits) of the fatty acid fraction of the oil is made up of medium chainfatty acids, i.e., fatty acids with aliphatic tails (i.e., carbonchains) having 6 to 14 carbons. In some embodiments, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 85%, about 90% or about 95% ofthe fatty acid fraction of the oil is made up of medium chain fattyacids. As used herein, “predominantly” means that greater than or equalto 50% of the fatty acid fraction of the oil is made up of medium-chainfatty acids, i.e., fatty acids with aliphatic carbon chains having 6 to14 carbon atoms. Those of skill in the art that will readily appreciatethat the terms “alkyl content” or “alkyl distribution” of an oil can beused in place of the term “fatty acid fraction” of an oil incharacterizing a given oil or solubilizing agent, and these terms areused interchangeable herein. As such, medium chain oils suitable for usein the formulations disclosed herein include medium chain oils whereinthe fatty acid fraction of the oil is substantially medium chain fattyacids, or medium chain oils wherein the alkyl content or alkyldistribution of the oil is substantially medium chain alkyls (C6-C12alkyls). It will be understood by those of skill in the art that themedium chain oils suitable for use in the formulations disclosed hereinare pharmaceutical grade (e.g., pharmaceutical grade medium chain oils).Examples of medium chain oils include, for example and withoutlimitation, medium chain fatty acids, medium chain fatty acid esters ofglycerol (e.g., for example, mono-, di-, and triglycerides), mediumchain fatty acid esters of propylene glycol, medium chain fatty acidderivatives of polyethylene glycol, and combinations thereof.

The term “ECN” or “equivalent carbon number” means the sum of the numberof carbon atoms in the fatty acid chains of an oil, and can be used tocharacterize an oil as, for example, a medium chain oil or a long-chainoil. For example, tripalmitin (tripalmitic glycerol), which is a simpletriglyceride containing three fatty acid chains of 16 carbon atoms, hasan ECN of 3×16=48. Conversely, a triglyceride with an ECN=40 may have“mixed” fatty acid chain lengths of 8, 16 and 16; 10, 14 and 16; 8, 14and 18; etc. Naturally occurring oils are frequently “mixed” withrespect to specific fatty acids, but tend not to contain both long chainfatty acids and medium chain fatty acids in the same glycerol backbone.Thus, triglycerides with ECN's of 21-42 typically contain predominantlymedium chain fatty acids; while triglycerides with ECN's of greater than43 typically contain predominantly long chain fatty acids. For example,the ECN of corn oil triglyceride in the USP would be in the range of51-54. Medium chain diglycerides with ECN's of 12-28 will often containpredominantly medium chain fatty chains, while diglycerides with ECN'sof 32 or greater will typically contain predominantly long chain fattyacid tails. Monoglycerides will have an ECN that matches the chainlength of the sole fatty acid chain. Thus, monoglyceride ECN's in therange of 6-14 contain mainly medium chain fatty acids, andmonoglycerides with ECN's 16 or greater will contain mainly long chainfatty acids.

The average ECN of a medium chain triglyceride oil is typically 21-42.For example, as listed in the US Pharmacopeia (USP), medium chaintriglycerides have the following composition as the exemplary oil setforth in the table below:

Fatty-acid Tail Length % of oil Exemplary Oil 6 ≤2.0 2.0 8 50.0-80.070.0 10 20.0-50.0 25.0 12 ≤3.0 2.0 14 ≤1.0 1.0and would have an average ECN of3*[(6*0.02)+(8*0.70)+(10*0.25)+(12*0.02)+(14*0.01)]=25.8. The ECN of theexemplary medium chain triglycerides oil can also be expressed as arange (per the ranges set forth in the USP) of 24.9-27.0. For oils thathave mixed mono-, di-, and triglycerides, or single and double fattyacid glycols, the ECN of the entire oil can be determined by calculatingthe ECN of each individual component (e.g., C8 monoglycerides, C8diglycerides, C10 monoglycerides, and C10 monoglycerides) and taking thesum of the relative percentage of the component multiplied by the ECNnormalized to a monoglyceride for each component. For example, the oilhaving C8 and C10 mono- and diglycerides shown in the table below has anECN of 8.3, and is thus a medium chain oil.

ECN as % of oil ECN as % of oil Fatty-acid Chain (chain length) ×normalized to Length % of oil (% in oil) monoglyceride C8 monoglyceride47 8 × 0.47 = 3.76  3.76 C10 monoglyceride 8 10 × 0.08 = 0.8 0.8 C8diglyceride 38 2 × (8 × 0.38) = 6.08 6.08/2 = 3.04 C10 diglyceride 7 2 ×(10 × 0.07) = 1.4 1.4/2 = 0.7 OIL ECN 8.3 (normalized to monoglycerides)

Expressed differently, ECN can be calculated as each chain length in thecomposition multiplied by its relative percentage in the oil:(8*0.85)+(10*0.15)=8.3.

The term “excipients,” as used herein, refers to non-API ingredientssuch as solubilizing agents, anti-oxidants, oils, lubricants, and othersused in formulating pharmaceutical products.

The term “patient” or “subject” refers to an individual to whom thepharmaceutical composition is administered.

The term “pharmaceutical composition” refers to a pharmaceuticalcomposition comprising at least a solubilizing agent and estradiol. Asused herein, pharmaceutical compositions are delivered, for example viasuppository (i.e., vaginal suppository), or absorbed vaginally.

The term “progestin” means any natural or man-made substance that haspharmacological properties similar to progesterone.

The terms “treat,” “treating,” and “treatment” refer to any indicia ofsuccess in the treatment or amelioration of an injury, disease, orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,disease, or condition more tolerable to the patient; slowing in the rateof degeneration or decline; or improving a patient's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subject parameters, including the results of a physicalexamination, neuropsychiatric examinations, or psychiatric evaluation.

The terms “atrophic vaginitis,” “vulvovaginal atrophy,” “vaginalatrophy,” and “VVA” are used herein interchangeably. The molecularmorphology of VVA is well known in the medical field.

As used herein, “sexual dysfunction” refers to a condition having one ormore symptoms of difficulty during any one or more stages. Thedysfunction can prevent an individual from enjoying sexual activity.Non-limiting examples of symptoms of sexual dysfunction include: reducedsexual desire, reduced sexual pleasure, reduced sexual arousal andexcitement, aversion to and avoidance of genital sexual contact,inability to attain or maintain arousal, and persistent or recurrentdelay of, or absence of orgasm. Sexual dysfunction may be lifelong (noeffective performance ever) or acquired (after a period of normalfunction); generalized or limited to certain situations or certainpartners; and total or partial.

As used herein, “sexual desire” refers to the frequency of wanting toengage in sexual activity and/or the frequency of engaging in sexualactivity as perceived by the individual. Sexual desire can be expressed,for example, in one or more cognitive activities, including thefrequency of sexual thoughts, the extent of enjoyment of movies, books,music, etc. having sexual content and/or the extent of enjoyment orpleasure of thinking and fantasizing about sex as perceived by theindividual.

As used herein, “sexual arousal” refers to the frequency of becomingsexually aroused, how readily sexual arousal occurs and/or if arousal ismaintained, as perceived by the individual. Psychologically, arousal caninclude factors such as increased desire for sexual activity andexcitement related to sexual activity. Physiologically, arousal caninclude increased blood flow to the genitals, causing clitoralengorgement, as well as vaginal lubrication.

As used herein, “lubrication” refers to wetness in and around the vaginabefore, during, or after sexual activity. Increasing lubrication caninclude increasing the frequency of lubrication; decreasing thedifficulty of becoming lubricated; and/or decreasing the difficulty inmaintaining lubrication.

As used herein, “satisfaction” refers to one or more positive emotions(e.g., contentment, fulfillment, gratification, and the like) related toa sexual activity or sexual relationship. Satisfaction can include, forexample, satisfaction with occurrence of sexual arousal or orgasm,satisfaction with the amount of closeness with a partner, andsatisfaction with overall sex life.

As used herein, “orgasm” refers to the highest point of sexualexcitement characterized by a subjective experience of intense pleasuremarked normally by vaginal contractions in females. Increasing orgasmcan include increasing the frequency, duration, and/or intensity oforgasms in a subject. Increasing orgasm can also include decreasing thedifficulty of reaching orgasm.

II. INTRODUCTION

Provided herein are pharmaceutical compositions comprising solubilizedestradiol designed to be absorbed vaginally. The pharmaceuticalcompositions disclosed herein are designed to be absorbed and have theirtherapeutic effect locally, e.g., in vaginal or surrounding tissue.Further disclosed herein are data demonstrating efficacy of thepharmaceutical compositions disclosed, as well as methods relating tothe pharmaceutical compositions. Generally, the pharmaceuticalcompositions disclosed herein are useful in VVA, dyspareunia, and otherindications caused by decrease or lack of estrogen.

Additional aspects and embodiments of this disclosure include: providingincreased patient ease of use while potentially minimizing certain sideeffects from inappropriate insertion, minimizing incidence ofvulvovaginal mycotic infection compared to incidence of vulvovaginalmycotic infection due to usage of other vaginally applied estradiolproducts; and, improved side effect profile (e.g., pruritus) comparedto, for example, VAGIFEM® (estradiol vaginal tablets, Novo Nordisk;Princeton, N.J.).

III. PHARMACEUTICAL COMPOSITIONS

Functionality

According to embodiments, the pharmaceutical compositions disclosedherein are alcohol-free or substantially alcohol-free. Thepharmaceutical compositions offer provide for improved patientcompliance because of improvements over the prior offering. According toembodiments, the pharmaceutical compositions disclosed herein areencapsulated in soft gelatin capsules, which improve comfort during use.According to embodiments, the pharmaceutical compositions aresubstantially liquid, which are more readily absorbed in the vaginaltissue, and also are dispersed over a larger surface area of the vaginaltissue.

Estradiol

According to embodiments, the pharmaceutical compositions disclosedherein are for vaginal insertion in a single or multiple unit dosageform. According to embodiments, the estradiol in the pharmaceuticalcompositions is at least about: 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% solubilized. According to embodiments and wherethe estradiol is not 100% solubilized, the remaining estradiol ispresent in a micronized (crystalline) form that is absorbable by thebody and retains biological functionality, either in its micronized formor in another form which the micronized form is converted to afteradministration.

According to embodiments, all or some of the estradiol is solubilized ina solubilizing agent during manufacturing process. According toembodiments, all or some of the estradiol is solubilized followingadministration (e.g., the micronized portion where the estradiol is not100% solubilized is solubilized in a body fluid after administration).According to embodiments, because the estradiol is solubilized, thesolubilizing agents taught herein, with or without additional excipientsother than the solubilizing agents, are liquid or semi-solid. To theextent the estradiol is not fully solubilized at the time ofadministration/insertion, the estradiol should be substantiallysolubilized at a body temperature (average of 37° C.) and, generally, atthe pH of the vagina (ranges from 3.8 to 4.5 in healthy patients; or 4.6to 6.5 in VVA patients).

According to embodiments, the estradiol can be added to thepharmaceutical compositions disclosed herein as estradiol, estradiolhemihydrate, or other grade estradiol forms used in pharmaceuticalcompositions or formulations.

According to embodiments, estradiol dosage strengths vary. Estradiol (orestradiol hemihydrate, for example, to the extent the water content ofthe estradiol hemihydrate is accounted for) dosage strength of is fromat least about 1 microgram (μg or μg) to at least about 50 μg. Specificdosage embodiments contain at least about: 1 μg, 2 μg, 3 μg, 4 μg, 5 μg,6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg,17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, 25 μg, 26 μg, 27μg, 28 μg, 29 μg, 30 μg, 31 μg, 32 μg, 33 μg, 34 μg, 35 μg, 36 μg, 37μg, 38 μg, 39 μg, 40 μg, 41 μg, 42 μg, 43 μg, 44 μg, 45 μg, 46 μg, 47μg, 48 μg, 49 μg, or 50 μg estradiol. According to embodiments, thepharmaceutical compositions contain at least about 2.5 μg; 4 μg 6.25 μg,7.5 μg, 12.5 μg, 18.75 μg of estradiol. According to embodiments, thepharmaceutical compositions contain from about 1 μg to about 10 μg, from3 μg to 7 μg, from about 7.5 μg to 12.5 μg, from about 10 μg to about 25μg, about 1 μg, about 2.5 μg, from about 23.5 μg to 27.5 μg, from about7.5 μg to 22.5 μg, from 10 μg to 25 μg of estradiol. The lowestclinically effective dose of estradiol is used for treatment of VVA andother indications set forth herein. In some embodiments, the estradioldosage is about 4 μg. In one embodiment, the estradiol dosage is about10 μg. In another embodiment, the estradiol dosage is about 25 μg.

Solvent System

According to embodiments, the solvent system that solubilizes theestradiol are medium chain fatty acid based solvents, together withother excipients. According to embodiments, the solvent system includesnon-toxic, pharmaceutically acceptable solvents, co-solvents,surfactants, and other excipients suitable for vaginal delivery orabsorption.

According to embodiments, oils having medium chain fatty acids as amajority component are used as solubilizing agents to solubilizeestradiol. According to embodiments, the solubilizing agents comprisemedium chain fatty acid esters (e.g., esters of glycerol, ethyleneglycol, or propylene glycol) or mixtures thereof. According toembodiments, the medium chain fatty acids comprise chain lengths from C6to C14. According to embodiments the medium chain fatty acids comprisechain lengths from C6 to C12. According to embodiments the medium chainfatty acids substantially comprise chain lengths from C8-C10. ECN's formedium chain oils will be in the range of 21-42 for triglycerides, 12-28for diglycerides, and 6-14 for monoglycerides.

According to embodiments, the medium chain fatty acids are saturated.According to embodiments, the medium chain fatty acids are predominantlysaturated, i.e., greater than about 60% or greater than about 75%saturated.

According to embodiments, estradiol is soluble in the solubilizing agentat room temperature, although it may be desirable to warm certainsolubilizing agents during manufacture to improve viscosity. Accordingto embodiments, the solubilizing agent is liquid at between roomtemperature and about 50° C., at or below 50° C., at or below 40° C., orat or below 30° C.

According to embodiments, the solubility of estradiol in the mediumchain oil, medium chain fatty acid, or solubilizing agent (oroil/surfactant) is at least about 0.01 wt %, 0.02 wt %, 0.05 wt %, 0.06wt %, 0.08 wt %, 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, 1.0 wt %, or higher.

According to embodiments, medium chain solubilizing agents include, forexample and without limitation saturated medium chain fatty acids:caproic acid (C6), enanthic acid (C7), caprylic acid (C8), pelargonicacid (C9), capric acid (C10), undecylic acid(C11), lauric acid (C12),tridecylic acid (C13), or myristic acid (C14). According to embodiments,the solubilizing agent includes oils made of these free medium chainfatty acids, oils of medium chain fatty acid esters of glycerin,propylene glycol, or ethylene glycol, or combinations thereof. Theseexamples comprise predominantly saturated medium chain fatty acids(i.e., greater than 50% of the fatty acids are medium chain saturatedfatty acids). According to embodiments, predominantly C6 to C12saturated fatty acids are contemplated. According to embodiments, thesolubilizing agent is selected from at least one of a solvent orco-solvent.

According to embodiments, glycerin based solubilizing agents include:mono-, di-, or triglycerides and combinations and derivatives thereof.Exemplary glycerin based solubilizing agents include MIGLYOLs®, whichare caprylic/capric triglycerides (SASOL Germany GMBH, Hamburg).MIGLYOLs includes MIGLYOL 810 (caprylic/capric triglyceride), MIGLYOL812 (caprylic/capric triglyceride), MIGLYOL 816 (caprylic/caprictriglyceride), and MIGLYOL 829 (caprylic/capric/succinic triglyceride).Other caprylic/capric triglyceride solubilizing agents are likewisecontemplated, including, for example: caproic/caprylic/capric/laurictriglycerides; caprylic/capric/linoleic triglycerides;caprylic/capric/succinic triglycerides. According to embodiments, CAPMULMCM, medium chain mono- and di-glycerides, is the solubilizing agent.Other and triglycerides of fractionated vegetable fatty acids, andcombinations or derivatives thereof can be the solubilizing agent,according to embodiments. For example, the solubilizing agent can be1,2,3-propanetriol (glycerol, glycerin, glycerine) esters of saturatedcoconut and palm kernel oil and derivatives thereof.

Ethylene and propylene glycols (which include polyethylene andpolypropylene glycols) solubilizing agents include: glyceryl mono- anddi-caprylates; propylene glycol monocaprylate (e.g., CAPMUL® PG-8 (theCAPMUL brands are owned by ABITEC, Columbus, Ohio)); propylene glycolmonocaprate (e.g., CAPMUL PG-10); propylene glycol mono- anddicaprylates; propylene glycol mono- and dicaprate; diethylene glycolmono ester (e.g., TRANSCUTOL®, 2-(2-ethoxyethoxy)ethanol, GATTEFOSSESAS); and diethylene glycol monoethyl ether. Other combinations of mono-and di-esters of propylene glycol or ethylene glycol are expresslycontemplated are the solubilizing agent.

According to embodiments, the solubilizing agent includes combinationsof mono- and di-propylene and ethylene glycols and mono-, di-, andtriglyceride combinations. According to embodiments, polyethylene glycolglyceride (GELUCIRE®, GATTEFOSSÉ SAS, Saint-Priest, France) can be usedherein as the solubilizing agent or as a surfactant. For example,GELUCIRE 44/14 (PEG-32 glyceryl laurate EP), a medium chain fatty acidesters of polyethylene glycol, is a polyethylene glycol glyceridecomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

According to embodiments, commercially available fatty acid glycerol andglycol ester solubilizing agents are often prepared from natural oilsand therefore may comprise components in addition to the fatty acidesters that predominantly comprise and characterize the solubilizingagent. Such other components may be, e.g., other fatty acid mono-, di-,and triglycerides; fatty acid mono- and diester ethylene or propyleneglycols, free glycerols or glycols, or free fatty acids, for example. Insome embodiments, when an oil/solubilizing agent is described herein asa saturated C₈ fatty acid mono- or diester of glycerol, the predominantcomponent of the oil, i.e., >50 wt % (e.g., >75 wt %, >85 wt % or >90 wt%) is caprylic monoglycerides and caprylic diglycerides. For example,the Technical Data Sheet by ABITEC for CAPMUL MCM C8 describes CAPMULMCM C8 as being composed of mono and diglycerides of medium chain fattyacids (mainly caprylic) and describes the alkyl content as ≤1% C6, ≥95%C8, ≤5% C10, and ≤1.5% C12 and higher.

For example, MIGLYOL 812 is a solubilizing agent that is generallydescribed as a C8-C10 triglyceride because the fatty acid composition isat least about 80% triglyceride esters of caprylic acid (C8) and capricacid (C10). However, it also includes small amounts of other fattyacids, e.g., less than about 5% of caproic acid (C6), lauric acid (C12),and myristic acid (C14). The product information sheet for variousMIGLYOLs illustrate the various fatty acid components as follows:

Tests 810 812 818 829 840 Caproic acid (C6:0) max. 2.0 max. 2.0 max. 2max. 2 max. 2 Caprylic acid (C8:0) 65.0-80.0 50.0-65.0 45-65 45-55 65-80Capric acid (C10:0) 20.0-35.0 30.0-45.0 30-45 30-40 20-35 Lauric acid(C12:0) max. 2 max. 2 max. 3 max. 3 max. 2 Myristic acid (C14:0) max.1.0 max. 1.0 max. 1 max. 1 max. 1 Linoleic acid (C18:2) — — 2-5 — —Succinic acid — — — 15-20 — ECN 25.5-26.4 26.1-27  26.52-28.56  26-27.625.5-26.4

According to embodiments, anionic or non-ionic surfactants may be usedin pharmaceutical compositions containing solubilized estradiol. Ratiosof solubilizing agent(s) to surfactant(s) vary depending upon therespective solubilizing agent(s) and the respective surfactant(s) andthe desired physical characteristics of the resultant pharmaceuticalcomposition. For example and without limitation, CAPMUL MCM and anon-ionic surfactant may be used at ratios including 65:35, 70:30,75:25, 80:20, 85:15 and 90:10. Other non-limiting examples include:CAPMUL MCM and GELUCIRE 39/01 used in ratios including, for example andwithout limitation, 6:4, 7:3, and 8:2; CAPMUL MCM and GELUCIRE 43/01used in ratios including, for example and without limitation, 7:3, and8:2; CAPMUL MCM and GELUCIRE 50/13 used in ratios including, for exampleand without limitation, 7:3, and 8:2, and 9:1.

Other Excipients

According to embodiments, the pharmaceutical composition furtherincludes a surfactant. The surfactant can be a nonionic surfactant,cationic surfactant, anionic surfactant, or mixtures thereof. Suitablesurfactants include, for example, water-insoluble surfactants having ahydrophilic-lipophilic balance (HLB) value less than 12 andwater-soluble surfactants having a HLB value greater than 12.Surfactants that have a high HLB and hydrophilicity, aid the formationof oil-water droplets. The surfactants are amphiphilic in nature and arecapable of dissolving or solubilizing relatively high amounts ofhydrophobic drug compounds.

Non-limiting examples, include, Tween, Dimethylacetamide (DMA), Dimethylsulfoxide (DMSO), Ethanol, Glycerin, N-methyl-2-pyrrolidone (NMP), PEG300, PEG 400, Poloxamer 407, Propylene glycol, Phospholipids,Hydrogenated soy phosphatidylcholine (HSPC),Distearoylphosphatidylglycerol (DSPG),L-α-dimyristoylphosphatidylcholine (DMPC),L-α-dimyristoylphosphatidylglycerol (DMPG), Polyoxyl 35 castor oil(CREMOPHOR EL, CREMOPHOR ELP), Polyoxyl 40 hydrogenated castor oil(Cremophor RH 40), Polyoxyl 60 hydrogenated castor oil (CREMOPHOR RH60), Polysorbate 20 (TWEEN 20), Polysorbate 80 (TWEEN 80),d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Solutol HS-15,Sorbitan monooleate (SPAN 20), PEG 300 caprylic/capric glycerides(SOFTIGEN 767), PEG 400 caprylic/capric glycerides (LABRASOL), PEG 300oleic glycerides (LABRAFIL M-1944CS), Polyoxyl 35 Castor oil (ETOCAS35), Glyceryl Caprylate (Mono- and Diglycerides) (IMWITOR), PEG 300linoleic glycerides (LABRAFIL M-2125CS), Polyoxyl 8 stearate (PEG 400monosterate), Polyoxyl 40 stearate (PEG 1750 monosterate), andcombinations thereof. Additionally, suitable surfactants include, forexample, polyoxyethylene derivative of sorbitan monolaurate such aspolysorbate, caprylcaproyl macrogol glycerides, polyglycolyzedglycerides, and the like.

According to embodiments, the non-ionic surfactant is selected from oneor more of glycerol and polyethylene glycol esters of long chain fattyacids, for example, lauroyl macrogol-32 glycerides or lauroylpolyoxyl-32 glycerides, commercially available as GELUCIRE, including,for example, GELUCIRE 39/01 (glycerol esters of saturated C12-C18 fattyacids), GELUCIRE 43/01 (hard fat NF/JPE) and GELUCIRE 50/13 (stearoylmacrogol-32 glycerides EP, stearoyl polyoxyl-32 glycerides NF, stearoylpolyoxylglycerides (USA FDA IIG)). These surfactants may be used atconcentrations greater than about 0.01%, and typically in variousamounts of about 0.01%-10.0%, 10.1%-20%, and 20.1%-30%. In someembodiments, surfactants may be used at concentrations of about 1% toabout 10% (e.g., about 1% to about 5%, about 2% to about 4%, about 3% toabout 8%).

According to embodiments, non-ionic surfactants include, for example andwithout limitation: one or more of oleic acid, linoleic acid, palmiticacid, and stearic acid. According to embodiments, non-ionic surfactantscomprise polyethylene sorbitol esters, including polysorbate 80, whichis commercially available under the trademark TWEEN® 80 (polysorbate 80)(Sigma Aldrich, St. Louis, Mo.). Polysorbate 80 includes approximately60%-70% oleic acid with the remainder comprising primarily linoleicacids, palmitic acids, and stearic acids. Polysorbate 80 may be used inamounts ranging from about 5 to 50%, and according to embodiments, about30% of the pharmaceutical composition total mass.

According to embodiments, the non-ionic surfactant includes PEG-6palmitostearate and ethylene glycol palmitostearate, which are availablecommercially as TEFOSE® 63 (GATTEFOSSE SAS, Saint-Priest, France), whichcan be used with, for example, CAPMUL MCM having ratios of MCM to TEFOSE63 of, for example, 8:2 or 9:1. According to embodiments, othersolubilizing agents/non-ionic surfactants combinations include, forexample, MIGLYOL 812:GELUCIRE 50/13 or MIGLYOL 812:TEFOSE 63.

According to embodiments, the surfactant can be an anionic surfactant,for example: ammonium lauryl sulfate, dioctyl sodium sulfosuccinate,perfluoro-octane sulfonic acid, potassium lauryl sulfate, or sodiumstearate. Cationic surfactants are also contemplated.

According to embodiments, non-ionic or anionic surfactants can be usedalone with at least one solubilizing agent or can be used in combinationwith other surfactants. Accordingly, such surfactants, or any otherexcipient as set forth herein, may be used to solubilize estradiol. Thecombination of solubilizing agent, surfactant, and other excipientsshould be designed whereby the estradiol is absorbed into the vaginaltissue. According to embodiments, the pharmaceutical composition willresult in minimal vaginal discharge.

According to embodiments, the pharmaceutical composition furtherincludes at least one thickening agent. Generally, a thickening agent isadded when the viscosity of the pharmaceutical composition results lessthan desirable absorption. According to embodiments, the surfactant(s)disclosed herein may also provide thickening of the pharmaceuticalcomposition that, upon release, will aid the estradiol in being absorbedby the vaginal mucosa while minimizing vaginal discharge. Examples ofthickening agents include: hard fats; propylene glycol; a mixture ofhard fat EP/NF/JPE, glyceryl ricinoleate, ethoxylated fatty alcohols(ceteth-20, steareth-20) EP/NF (available as OVUCIRE® 3460, GATTEFOSSE,Saint-Priest, France); a mixture of hard fat EP/NF/JPE, glycerolmonooleate (type 40) EP/NF (OVUCIRE WL 3264; a mixture of hard fatEP/NF/JPE, glyceryl monooleate (type 40) EP/NF (OVUCIRE WL 2944); anon-ionic surfactant comprising PEG-6 stearate, ethylene glycolpalmitostearate, and PEG-32 stearate; TEFOSE 63 or a similar product;and a mixture of various hard fats (WITEPSOL®' Sasol Germany GmbH,Hamburg, Germany). Other thickening agents such as the alginates,certain gums such as xanthan gums, agar-agar, iota carrageenans, kappacarrageenans, etc. Several other compounds can act as thickening agentslike gelatin, and polymers like HPMC, PVC, and CMC. According toembodiments, the viscosity of pharmaceutical compositions in accordancewith various embodiments may comprise from about 50 cps to about 1000cps at 25° C. A person of ordinary skill in the art will readilyunderstand and select from suitable thickening agents.

According to embodiments, the thickening agent is a non-ionicsurfactant. For example, polyethylene glycol saturated or unsaturatedfatty acid ester or diester is the non-ionic surfactant thickeningagent. In embodiments, the non-ionic surfactant includes a polyethyleneglycol long chain (C16-C20) fatty acid ester and further includes anethylene glycol long chain fatty acid ester, such as PEG-fatty acidesters or diesters of saturated or unsaturated C16-C18 fatty acids,e.g., oleic, lauric, palmitic, and stearic acids. In embodiments, thenon-ionic surfactant includes a polyethylene glycol long chain saturatedfatty acid ester and further includes an ethylene glycol long chainsaturated fatty acid ester, such as PEG- and ethylene glycol-fatty acidesters of saturated C16-C18 fatty acids, e.g., palmitic and stearicacids. Such non-ionic surfactant can comprise PEG-6 stearate, ethyleneglycol palmitostearate, and PEG-32 stearate, such as but not limited toTEFOSE 63.

According to embodiments, TEFOSE 63 is used to provide additionalviscosity and/or spreadability in the vagina so as to retard flow of thecomposition out of the vagina. While the pharmaceutical compositionremains liquid, the viscosity of such a pharmaceutical compositioncauses the liquid to remain in the API absorption area whereby thepharmaceutical composition is substantially absorbed by the tissue.Suprisingly, the addition of an excipient to increase the viscosityand/or spreadability of the pharmaceutical compositions herein allowsthe administration of a pharmaceutical composition that is liquid atbody temperature but does not excessively discharge from the vagina whenthe patient is standing, which allows the patients to be ambulatoryafter administration of the pharmaceutical compositions.

According to embodiments, the non-ionic surfactant used as a thickeningagent is not hydrophilic and has good emulsion properties. Anillustrative example of such surfactant is TEFOSE 63, which has ahydrophilic-lipophilic balance (HLB) value of about 9-10.

According to embodiments, the pharmaceutical composition furtherincludes one or more mucoadherent agents to improve vaginal absorptionof the estradiol by, for example, increasing the viscosity of of thepharmaceutical composition whereby flow out of the vagina is retarded.According to other embodiments, alone or in addition to changes inviscosity, the mucoadhesive agent causes the pharmaceutical compositionto adhere to the vaginal tissue chemically or mechanically. For example,a mucoadherent agent can be present to aid the pharmaceuticalcomposition with adherence to the mucosa upon activation with water.According to embodiments, polycarbophil is the mucoadherent agent.According to embodiments, other mucoadherent agents include, for exampleand without limitation: poly (ethylene oxide) polymers having amolecular weight of from about 100,000 to about 900,000; chitosans;carbopols including polymers of acrylic acid crosslinked with allylsucrose or allyl pentaerythritol; polymers of acrylic acid and C10-C30alkyl acrylate crosslinked with allyl pentaerythritol; carbomerhomopolymer or copolymer that contains a block copolymer of polyethyleneglycol and a long chain alkyl acid ester; and the like. According toembodiments, various hydrophilic polymers and hydrogels may be used asthe mucoadherent agent. According to certain embodiments, the polymersor hydrogels can swell in response to contact with vaginal tissue orsecretions, enhancing moisturizing and mucoadherent effects. Theselection and amount of hydrophilic polymer may be based on theselection and amount of solubilizing agent. In some embodiments, thepharmaceutical composition includes a hydrophilic polymer but optionallyexcludes a gelling agent. In embodiments having a hydrogel, from about5% to about 10% of the total mass may comprise the hydrophilic polymer.In further embodiments, hydrogels may be employed. A hydrogel maycomprise chitosan, which swell in response to contact with water. Invarious embodiments, a cream pharmaceutical composition may comprisePEG-90M. In some embodiments, a mucoadherent agent is present in thepharmaceutical formulation, in the soft gel capsule, or both.

According to embodiments, the pharmaceutical compositions include one ormore thermoreversible gels, typically of the hydrophilic natureincluding for example and without limitation, hydrophilic sucrose andother saccharide-based monomers (U.S. Pat. No. 6,018,033, which isincorporated by reference).

According to embodiments, the pharmaceutical composition furtherincludes a lubricant. In some embodiments, a lubricant can be present toaid in formulation of a dosage form. For example, a lubricant may beadded to ensure that capsules or tablets do not stick to one anotherduring processing or upon storage. Any suitable lubricant may be used.For example, lecithin, which is a mixture of phospholipids, is thelubricant.

According to embodiments, the pharmaceutical composition furtherincludes an antioxidant. Any suitable anti-oxidant may be used. Forexample, butylated hydroxytoluene, butylated hydroxyanisole, and VitaminE TPGS.

According to embodiments, the pharmaceutical composition includes about20% to about 80% solubilizing agent by weight, about 0.1% to about 5%lubricant by weight, and about 0.01% to about 0.1% antioxidant byweight.

The choice of excipient will depend on factors such as, for example, theeffect of the excipient on solubility and stability. Additionalexcipients used in various embodiments may include colorants andpreservatives. Examples of colorants include FD&C colors (e.g., blue No.1 and Red No. 40), D&C colors (e.g., Yellow No. 10), and opacifiers(e.g., Titanium dioxide). According to embodiments, colorants, compriseabout 0.1% to about 2% of the pharmaceutical composition by weight.According to embodiments, preservatives in the pharmaceuticalcomposition comprise methyl and propyl paraben, in a ratio of about10:1, and at a proportion of about 0.005% and 0.05% by weight.

Generally, the solubilizing agents, excipients, other additives used inthe pharmaceutical compositions described herein, are non-toxic,pharmaceutically acceptable, compatible with each other, and maintainstability of the pharmaceutical composition and the various componentswith respect to each other. Additionally, the combination of variouscomponents that comprise the pharmaceutical compositions will maintainwill result in the desired therapeutic effect when administered to asubject.

Solubility of Estradiol

According to embodiments, solubilizing agents comprising mixtures ofmedium chain fatty acid glycerides, e.g., C₆-C12, C₈-C12, or C₈-C10fatty acid mono- and diglycerides or mono-, di-, and triglyceridesdissolve estradiol. As illustrated in the Examples, good results wereobtained with solubilizing agents that are predominantly a mixture ofC8-C10 saturated fatty acid mono- and diglycerides, or medium chaintriglycerides (e.g., MIGLYOL 810 or 812). Longer chain glycerides appearto be not as well suited for dissolution of estradiol.

A solubilizing agent comprising propylene glycol monocaprylate (e.g.,CAPRYOL) and 2-(2-Ethoxyethoxy)ethanol (e.g., TRANSCUTOL) solubilizedestradiol well.

IV. MANUFACTURE OF THE PHARMACEUTICAL COMPOSITION

According to embodiments, the pharmaceutical composition is prepared viablending estradiol with a pharmaceutically acceptable solubilizingagent, including for example and without limitation, at least one mediumchain fatty acid such as medium chain fatty acids consisting of at leastone mono-, di-, or triglyceride, or derivatives thereof, or combinationsthereof. According to embodiments, the pharmaceutical composition alsoincludes at least one glycol or derivatives thereof or combinationsthereof or combinations of at least one glyceride and glycol. Theglycol(s) may be used as solubilizing agents or to adjust viscosity and,thus, may be considered thickening agents, as discussed further herein.Optionally added are other excipients including, for example and withoutlimitation, anti-oxidants, lubricants, and the like. According toembodiments, the pharmaceutical composition includes sufficientsolubilizing agent to fully solubilize the estradiol. It is expresslyunderstood, however, the other volumes of solubilizing agent can be useddepending on the level of estradiol solubilization desired. Persons ofordinary skill in the art will know and understand how to determine thevolume of solubilizing agent and other excipients depending on thedesired percent of estradiol to be solubilized in the pharmaceuticalcomposition.

In illustrative embodiments, GELUCIRE 44/14 (lauroyl macrogol-32glycerides EP, lauroyl polyoxyl-32 glycerides NF, lauroylpolyoxylglycerides (USA FDA IIG)) is heated to about 65° C. and CAPMULMCM is heated to about 40° C. to facilitate mixing of the oil andnon-ionic surfactant, although such heating is not necessary to dissolvethe estradiol.

Specific Examples disclosed herein provide additional principles andembodiments illustrating the manufactures of the pharmaceuticalcompositions disclosed herein.

V. DELIVERY VEHICLE

Generally, the pharmaceutical compositions described herein deliveredintravaginally inside of a delivery vehicle, for example a capsule.According to embodiments, the capsules are soft capsules made ofmaterials well known in the pharmaceutical arts, for example, gelatin.However, according to embodiments, the delivery vehicle is integral withthe pharmaceutical composition (i.e., the pharmaceutical composition isthe delivery vehicle). In such embodiments the pharmaceuticalcompositions is a gel, cream, ointment, tablet, or other preparationthat is directly applied and absorbed vaginally.

According to embodiments, the capsules do not contain one or more of thefollowing: a hydrophilic gel-forming bioadhesive agent, a lipophilicagent, a gelling agent for the lipophilic agent, and/or ahydrodispersible agent. According to embodiments, the capsules do notcontain a hydrophilic gel-forming bioadhesive agent selected from:carboxyvinylic acid, hydroxypropylcellulose, carboxymethylcellulose,gelatin, xanthan gum, guar gum, aluminum silicate, and mixtures thereof.According to embodiments, the capsules do not contain a lipophilic agentselected from: a liquid triglyceride, a solid triglyceride (with amelting point of about 35° C.), carnauba wax, cocoa butter, and mixturesthereof. According to embodiments, the capsules do not contain ahydrophobic colloidal silica gelling agent. According to embodiments,the capsules do not contain a hydrodispersible agent selected from:polyoxyethylene glycol, polyoxyethylene glycol 7-glyceryl-cocoate, andmixtures thereof In some embodiments, the estradiol is formulated as aliquid composition consisting of a therapeutically effective amount ofestradiol; a caprylic/capric triglyceride; and a non-ionic surfactantcomprising PEG-6 palmitostearate and ethylene glycol palmitostearate. Insuch embodiments, a hydrophilic gel-forming bioadhesive agent in theliquid composition. In some such embodiments, the liquid composition iscontained with a gelatin capsule as described herein. In some suchembodiments, the capsule comprises gelatin and optionally one or morefurther components selected from the group consisting of gelatin,hydrolyzed gelatin, sorbitol-sorbitan solution, water, glycerin,titanium dioxide, FD&C Red #40, ethanol, ethyl acetate, propyleneglycol, polyvinyl acetate phthalate, isopropyl alcohol, polyethyleneglycol, and ammonium hydroxide.

According to embodiments, the delivery vehicle is designed for ease ofinsertion. According to embodiments, the delivery vehicle is sizedwhereby it can be comfortably inserted into the vagina. According toembodiments, the delivery vehicle is prepared in a variety ofgeometries. For example, the delivery vehicle is shaped as a tear drop,a cone with frustoconical end, a cylinder, a cylinder with larger “cap”portion, or other shapes suitable for and that ease insertion into thevagina. According to embodiments, the delivery vehicle is used inconnection with an applicator. According to other embodiments, thedelivery vehicle is inserted digitally.

According to embodiments, a method for the treatment of VVA, includingdyspareunia, vaginal dryness, and estrogen-deficient urinary states(including urinary tract infections), is provided wherein a compositionfor the treatment of VVA is digitally insert approximately two inchesinto the vagina or in the third of the vagina closest to the opening ofthe vagina and results in at least one of: improved compliance comparedto other products for the treatment of VVA; improved user experiencecompared to other products for the treatment of VVA; and statisticallysignificantly improved symptoms of VVA, compared to placebo or baselinewithin one of two, four, six, eight, ten, or twelve or more weeks afterinitiation of administration. According to embodiments, a method for thetreatment of VVA, including dyspareunia, vaginal dryness, andestrogen-deficient urinary states (including urinary tract infections),is provided wherein a delivery vehicle containing a composition for thetreatment of VVA and a tear drop shape as disclosed herein is insertapproximately two inches into the vagina or in the third of the vaginaclosest to the opening of the vagina and results in at least one of:improved compliance compared to other products for the treatment of VVA;improved user experience compared to other products for the treatment ofVVA; and statistically significantly improved symptoms of VVA, comparedto placebo or baseline within one of two, four, six, eight, ten, ortwelve or more weeks after initiation of administration.

With reference to FIG. 2, delivery vehicle 200 includes pharmaceuticalcomposition 202 and capsule 204. Width 208 represents the thickness ofcapsule 204, for example about 0.108 inches. The distance from one endof delivery vehicle 200 to another is represented by distance 206, forexample about 0.690 inches. The size of delivery vehicle 200 may also bedescribed by the arc swept by a radius of a given length. For example,arc 210, which is defined by the exterior of gelatin 204, is an arcswept by a radius of about 0.189 inches. Arc 212, which is defined bythe interior of capsule 204, is an arc swept by a radius of about 0.0938inches. Arc 214, which is defined by the exterior of gelatin 204opposite arc 210, is an arc swept by a radius of about 0.108 inches.Suitable capsules of other dimensions may be provided. According toembodiments, capsule 204 has dimensions the same as or similar to theratios as provided above relative to each other. In some embodiment, thegelatin capsule further comprises one or more components selected fromthe group consisting of hydrolyzed gelatin, sorbitol-sorbitan solution,water, glycerin, titanium dioxide, FD&C Red #40, ethanol, ethyl acetate,propylene glycol, polyvinyl acetate phthalate, isopropyl alcohol,polyethylene glycol, and ammonium hydroxide.

According to embodiments, the delivery vehicle is designed to remainingin the vagina until the pharmaceutical compositions are released.According to embodiments, delivery vehicle dissolves intravaginally andis absorbed into the vaginal tissue with the pharmaceutical composition,which minimizes vaginal discharge. In such embodiments, deliverymechanism is made from constituents that are non-toxic, for example,gelatin.

Design Factors for Vaginally Inserted Pharmaceutical Compositions

According to embodiments, the pharmaceutical composition is designed tomaximize favorable characteristics that lead to patient compliance(patients that discontinue treatment prior to completion of theprescribed course of therapy), without sacrificing efficacy. Favorablecharacteristics include, for example, lack of or reduction of irritationrelative to other hormone replacement pessaries, lack of or reduction invaginal discharge of the pharmaceutical composition and delivery vehiclerelative to other hormone replacement pessaries, lack of or reduction ofpharmaceutical composition or delivery vehicle residue inside thevagina, ease of administration compared to other hormone replacementpessaries, or improved efficacy of drug product relative to otherwisesimilar pharmaceutical compositions.

According to embodiments, the pharmaceutical composition isnon-irritating or minimizes irritation. Patient irritation includespain, pruritus (itching), soreness, excessive discharge, swelling, orother similar conditions. Patient irritation results in poor compliance.Non-irritating or reduced irritation pharmaceutical compositions aremeasured relative to competing hormone pessaries, including tablets,creams, or other intravaginal estrogen delivery forms.

According to embodiments, the pharmaceutical compositions does notresult in systemic exposure (e.g., blood circulation of estradiol),which improves safety. According to other embodiments, thepharmaceutical compositions disclosed herein result in significantlyreduced systemic exposure (e.g., blood circulation of estradiol) whencompared to other vaginally administered drugs on the market for thetreatment of VVA.

In certain embodiments, the administration of the pharmaceuticalcomposition provides a mean concentration (C_(ave)) value below 20.6pg/mL on Day 1 of the treatment, and/or a C_(ave) value below 19.4 pg/mLon Day 14 of the treatment, and/or a C_(ave) value below 11.5 pg/mL onDay 83 of the treatment. In certain embodiments, the administration ofthe pharmaceutical composition provides a mean concentration (C_(ave))value below 10 pg/mL on Day 1 of the treatment, and/or a C_(ave) valuebelow 7.3 pg/mL on Day 14 of the treatment, and/or a C_(ave) value below5.5 pg/mL on Day 83 of the treatment.

According to embodiments, the pharmaceutical composition does not leaveresidue inside the vagina. Rather, the pharmaceutical composition anddelivery vehicle are substantially absorbed or dispersed withoutresulting in unabsorbed residue or unpleasant sensations of non-absorbedor non-dispersed drug product. Measurement of lack of residue isrelative to other vaginally inserted products or can be measuredobjectively with inspection of the vaginal tissues. For example, certainother vaginally inserted products contain starch which can result ingreater discharge from the vagina following administration than. In someembodiments, the pharmaceutical compositions provided herein provide alower amount, duration, or frequency of discharge followingadministration compared to other vaginally inserted products (e.g.,compressed tablets).

According to embodiments, the pharmaceutical composition improvesvaginal discharge compared to other pessaries, including pessaries thatdeliver hormones. Ideally, vaginal discharge is eliminated, minimized,or improved compared to competing products.

According to embodiments, the pharmaceutical compositions disclosedherein are inserted digitally. According to embodiments, thepharmaceutical compositions are digitally inserted approximately twoinches into the vagina without a need for an applicator. According toembodiments, the pharmaceutical compositions are designed to be alsoinserted with an applicator, if desired. According to some embodiments,because the site of VVA is in the proximal region of the vagina (towardsthe vaginal opening), the pharmaceutical compositions disclosed hereinare designed to be inserted in the proximal portion of the vagina.

Through extensive experimentation, various medium chain fatty acidesters of glycerol and propylene glycol demonstrated one or morefavorable characteristics for development as a human drug product.According to embodiments, the solubilizing agent was selected from atleast one of a solvent or co-solvent. Suitable solvents and co-solventsinclude any mono-, di- or triglyceride and glycols, and combinationsthereof.

According to embodiments, the pharmaceutical composition is deliveredvia a gelatin capsule delivery vehicle. According to these embodiments,the pharmaceutical composition is a liquid pharmaceutical composition.According to embodiments, the delivery vehicle is a soft capsule, forexample a soft gelatin capsule. Thus, the pharmaceutical composition ofsuch embodiments is encapsulated in the soft gelatin capsule or othersoft capsule.

According to embodiments, the pharmaceutical composition includesestradiol that is at least about 80% solubilized in a solubilizing agentcomprising one or more C6 to C14 medium chain fatty acid mono-, di-, ortriglycerides and, optionally, a thickening agent. According toembodiments, the pharmaceutical composition includes estradiol that isat least about 80% solubilized one or more C6 to C12 medium chain fattyacid mono-, di-, or triglycerides, e.g., one or more C6 to C14triglycerides, e.g., one or more C6 to C12 triglycerides, such as one ormore C8-C10 triglycerides. These embodiments specifically contemplatethe estradiol being at least 80% solubilized. These embodimentsspecifically contemplate the estradiol being at least 90% solubilized.These embodiments specifically contemplate the estradiol being at least95% solubilized. These embodiments specifically contemplate theestradiol being fully solubilized.

As noted above, liquid pharmaceutical compositions are liquid at roomtemperature or at body temperature. For example, in some embodiments, apharmaceutical composition provided herein is a liquid formulationcontained within a soft gel capsule. Gels, hard fats, or other solidforms that are not liquid at room or body temperature are less desirablein embodiments of the pharmaceutical composition that are liquid.

The thickening agent serves to increase viscosity, e.g., up to about10,000 cP (10,000 mPa-s), typically to no more than about 5000 cP, andmore typically to between about 50 and 1000 cP. In embodiments, thenon-ionic surfactant, e.g., GELUCIRE or TEFOSE, may be solid at roomtemperature and require melting to effectively mix with the solubilizingagent. However, in these embodiments, the resultant pharmaceuticalcomposition remains liquid, albeit with greater viscosity, not solid.

According to embodiments, the pharmaceutical composition includesestradiol, the medium chain solubilizing agent, and the thickening agentas the ingredients delivered via a soft capsule delivery vehicle. Otheringredients, e.g., colorants, antioxidants, preservatives, or otheringredients may be included as well. However, the addition of otheringredients should be in amounts that do not materially change thesolubility of the estradiol, the pharmacokinetics of the pharmaceuticalcomposition, or efficacy of the pharmaceutical composition. Otherfactors that should be considered when adjusting the ingredients of thepharmaceutical composition include the irritation, vaginal discharge,intravaginal residue, and other relevant factors, for example those thatwould lead to reduced patient compliance.

Other contemplated ingredients include: oils or fatty acid esters,lecithin, mucoadherent agents, gelling agents, dispersing agents, or thelike.

VI. METHODS

According to embodiments, the pharmaceutical compositions disclosedherein can be used for the treatment of VVA, including the treatment ofat least one VVA symptom including: vaginal dryness, vaginal or vulvarirritation or itching, dysuria, dyspareunia, and vaginal bleedingassociated with sexual activity, among others. According to embodimentsthe methods of treatment are generally applicable to females.

According to embodiments, the pharmaceutical compositions disclosedherein can be used for the treatment of estrogen-deficient urinarystates. According to embodiments, the pharmaceutical compositionsdisclosed herein can be used for the treatment of dyspareunia, orvaginal bleeding associated with sexual activity.

According to embodiments, treatment of the VVA, estrogen-deficienturinary states, and dyspareunia and vaginal bleeding associated withsexual activity occurs by administering the pharmaceutical compositionsintravaginally. According to embodiments where the delivery vehicle is acapsule, the patient obtains the capsule and inserts the capsule intothe vagina, where the capsule dissolves and the pharmaceuticalcomposition is released into the vagina where it is absorbed into thevaginal tissue. In some embodiments, the pharmaceutical composition iscompletely absorbed into the vaginal tissue. In some embodiments, thepharmaceutical composition is substantially absorbed into the vaginaltissue (e.g., at least about 80% by weight, at least about 85% byweight, at least about 90% by weight, at least about 95% by weight, atleast about 97% by weight, at least about 98% by weight, or at leastabout 99% by weight of the composition is absorbed). According toembodiments, the capsule is inserted about two inches into the vagina,however the depth of insertion is generally any depth that allows foradsorption of substantially all of the pharmaceutical composition.According to embodiments, the capsule can also be applied using anapplicator that deposits the capsule at an appropriate vaginal depth asdisclosed herein. According to embodiments, the capsule is insert intothe lower third of the vagina (i.e., the third closest to the vaginalopening). According to embodiments, the softgel capsule can be held withthe larger end between the fingers as shown in FIG. 26A.

The subject will select a position that is most comfortable (e.g., areclining position as shown in FIG. 26B or a standing position as shownin FIG. 26C), and the subject will insert the softgel into the lowerthird of the vagina with the smaller end up. The softgel capsule willdissolve rapidly. The softgel can be inserted at any time of day andnormal activities can be immediately resumed. According to embodiments,the same time of day for all insertions of of the softgel is used.

According to embodiments where the pharmaceutical composition is acream, gel, ointment, or other similar preparation, the pharmaceuticalcomposition is applied digitally, as is well known and understood in theart.

Upon release of the pharmaceutical composition in the vagina, estradiolis locally absorbed. For example, following administration of thesuppository to the proximal region of the vagina of a patient provides atherapeutically effective concentration of estradiol over 24 hours inthe proximal region of the vagina.

According to embodiments, the timing of administration of thepharmaceutical composition of this disclosure may be conducted by anysafe means as prescribed by an attending physician. According toembodiments, a patient will administer the pharmaceutical composition(e.g., a capsule) intravaginally each day for 14 days, then twice weeklythereafter. In some such embodiments, the doses administered during thetwice weekly dosing period are administered approximately 3-4 daysapart. Typically, doses administered during the twice weekly dosingperiod do not exceed more than twice in a seven day period.

According to embodiments, the pharmaceutical compositions are vaginallyadministered with co-administration of an orally administeredestrogen-based (or progestin-based or progestin- and estrogen-based)pharmaceutical drug product, or patch, cream, gel, spray, transdermaldelivery system or other parenterally-administered estrogen-basedpharmaceutical drug product, each of which can include natural,bio-similar, or synthetic or other derived estrogens or progestins.According to embodiments, modulation of circulating estrogen levelsprovided via the administration of the pharmaceutical compositionsdisclosed herein, if any, are not intended to be additive to anyco-administered estrogen product and its associated circulating bloodlevels. According to other embodiments, co-administrated estrogenproducts are intended to have an additive effect as would be determinedby the patient physician.

According to embodiments, a method for estrogenizing vaginal tissue isprovided. The method includes administration of a (i.e., a suppository)or dosage as described herein. Estrogenized vaginal tissue is typicallycharacterized by one or more of the following properties: the presenceclear secretions on vaginal walls; rogation and elasticity of thevaginal walls; intact vaginal epithelium; and pink tissue color. Incontrast, de-estrogenized vaginal is characterized by decreased orabsent secretions; smooth tissue with fewer or no rugae; bleeding of thevaginal surface; development of petechiae (i.e., pinpoint, round spotson the skin due to bleeding, appearing red, brown, or purple); and paleor transparent tissues. Accordingly, estrogenizing vaginal tissueaccording to the method disclosed herein can include, increasing thelevel of vaginal secretions in a subject; increasing the number ofvaginal rugae in the subject; and/or decreasing bleeding or petechiae inthe subject. According to embodiments, a method for estrogenizingvaginal tissue is provided, the method including administering asuppository so as to provide an estradiol C_(max) or AUC as describedherein. According to embodiments, a method for estrogenizing vaginaltissue is provided, the method including administering a suppository soas to provide an estrone C_(max) or AUC as described herein.

According to embodiments, a method for estrogenizing the labia majoraand labia minora (collectively “labia”) is provided as described herein.Generally, the pharmaceutical composition is inserted digitally into thevagina approximately two inches or inserted into the third of the vaginaclosest to the vaginal opening as shown in FIGS. 26A, 26B, and 26C. Thegelatin capsule containing the pharmaceutical composition dissolves,ruptures, or otherwise releases the pharmaceutical composition into thevagina, whereby the lower third of the vagina and labia are bothreestrogenized. According to some embodiments, the pharmaceuticalcomposition is a liquid that partially flows to the labia and directlyreestrogenizes the labia.

According to embodiments, a method for estrogenizing the vulva isprovided as described herein. Generally, the pharmaceutical compositionis inserted digitally into the vagina approximately two inches orinserted into the third of the vagina closest to the vaginal opening asshown in FIGS. 26A, 26B, and 26C. The gelatin capsule containing thepharmaceutical composition dissolves, ruptures, or otherwise releasesthe pharmaceutical composition into the vagina, whereby the lower thirdof the vagina and vulva are both reestrogenized. According to someembodiments, the pharmaceutical composition is a liquid that partiallyflows to the vulval tissue and directly reestrogenizes the vulva.

According to embodiments, a method for treating vaginal dryness isprovided. The method includes administration of a soft gel vaginalestradiol formulation (i.e., a suppository) or dosage as describedherein. Treating vaginal dryness according to the method disclosedherein can include, decreasing the severity of vaginal dryness by 1%,5%, 10%, 15%, 20%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 99%. The decrease in severity can be obtainedfollowing 2 weeks of treatment, or 6 weeks of treatment, or 8 weeks oftreatment, or 12 weeks of treatment. In some embodiments, vaginaldryness is assessed using a severity scale, ranging from 0 to 4 pointswherein 0 indicates no dryness, 1 indicates mild dryness, 2 indicatesmoderate dryness, and 3 indicates severe dryness.

In some embodiments, the method for treating vaginal dryness includesreducing the dryness severity score from 3, prior to treatment of asubject, to 2, after 2 weeks of treatment of the subject. In someembodiments, the method for treating vaginal dryness includes reducingthe dryness severity score from 2, prior to treatment of a subject, to1, after 2 weeks of treatment of the subject. In some embodiments, themethod for treating vaginal dryness includes reducing the drynessseverity score from 1, prior to treatment of subject, to 0, after 2weeks of treatment of the subject.

In some embodiments, the method for treating vaginal dryness includesreducing the dryness severity score from 3, prior to treatment of asubject, to 2, after 6 weeks of treatment of the subject. In someembodiments, the method for treating vaginal dryness includes reducingthe dryness severity score from 2, prior to treatment of a subject, to1, after 6 weeks of treatment of the subject. In some embodiments, themethod for treating vaginal dryness includes reducing the drynessseverity score from 1, prior to treatment of subject, to 0, after 6weeks of treatment of the subject.

In some embodiments, the method for treating vaginal dryness includesreducing the dryness severity score from 3, prior to treatment of asubject, to 2, after 8 weeks of treatment of the subject. In someembodiments, the method for treating vaginal dryness includes reducingthe dryness severity score from 2, prior to treatment of a subject, to1, after 8 weeks of treatment of the subject. In some embodiments, themethod for treating vaginal dryness includes reducing the drynessseverity score from 1, prior to treatment of subject, to 0, after 8weeks of treatment of the subject.

In some embodiments, the method for treating vaginal dryness includesreducing the dryness severity score from 3, prior to treatment of asubject, to 2, after 12 weeks of treatment of the subject. In someembodiments, the method for treating vaginal dryness includes reducingthe dryness severity score from 2, prior to treatment of a subject, to1, after 12 weeks of treatment of the subject. In some embodiments, themethod for treating vaginal dryness includes reducing the drynessseverity score from 1, prior to treatment of subject, to 0, after 12weeks of treatment of the subject.

In some embodiments, the method for treating vaginal dryness includesdecreasing the severity of dryness after two weeks of treatment, whereinthe severity is assessed on a scale of 0-3 points, and the averagedecrease ranges from a 0.5-point decrease to a 1.25-point decrease. Theaverage decrease can be determined by observing any suitable number ofsubjects. In some embodiments, the number of subjects is at least 100.In some embodiments, the number of subjects is at least 500. In someembodiments, the number of subjects ranges from 700 to 800. In someembodiments, the number of subjects ranges from 740 to 750. In someembodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vaginal dryness includesdecreasing the severity of dryness after six weeks of treatment, whereinthe severity is assessed on a scale of 0-3 points, and the averagedecrease ranges from a 0.75-point decrease to a 1.5-point decrease. Theaverage decrease can be determined by observing any suitable number ofsubjects. In some embodiments, the number of subjects is at least 100.In some embodiments, the number of subjects is at least 500. In someembodiments, the number of subjects ranges from 700 to 800. In someembodiments, the number of subjects ranges from 740 to 750. In someembodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vaginal dryness includesdecreasing the severity of dryness after eight weeks of treatment,wherein the severity is assessed on a scale of 0-3 points, and theaverage decrease ranges from a 0.9-point decrease to a 1.5-pointdecrease. The average decrease can be determined by observing anysuitable number of subjects. In some embodiments, the number of subjectsis at least 100. In some embodiments, the number of subjects is at least500. In some embodiments, the number of subjects ranges from 700 to 800.In some embodiments, the number of subjects ranges from 740 to 750. Insome embodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vaginal dryness includesdecreasing the severity of dryness after twelve weeks of treatment,wherein the severity is assessed on a scale of 0-3 points, and theaverage decrease ranges from a 0.9-point decrease to a 1.5-pointdecrease. The average decrease can be determined by observing anysuitable number of subjects. In some embodiments, the number of subjectsis at least 100. In some embodiments, the number of subjects is at least500. In some embodiments, the number of subjects ranges from 700 to 800.In some embodiments, the number of subjects ranges from 740 to 750. Insome embodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vaginal dryness includesadministering a suppository so as to provide an estradiol C_(max) or AUCas described herein. According to embodiments, a method for treatingvaginal dryness is provided, the method including administering asuppository so as to provide an estrone C_(max) or AUC as describedherein.

According to embodiments, a method for treating vulvar and/or vaginalitching or irritation is provided. The method includes administration ofa soft gel vaginal estradiol formulation (i.e., a suppository) or dosageas described herein. Treating vulvar and/or vaginal itching orirritation according to the method disclosed herein can include,decreasing the severity of vulvar and/or vaginal itching or irritationby 1%, 5%, 10%, 15%, 20%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 99%. The decrease in severity can be obtainedfollowing 2 weeks of treatment, or 6 weeks of treatment, or 8 weeks oftreatment, or 12 weeks of treatment. In some embodiments, vulvar and/orvaginal itching or irritation is assessed using a severity scale,ranging from 0 to 4 points wherein 0 indicates no itching or irritation,1 indicates mild itching or irritation, 2 indicates moderate itching orirritation, and 3 indicates severe itching or irritation.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes reducing the itching/irritation severityscore from 3, prior to treatment of a subject, to 2, after 2 weeks oftreatment of the subject. In some embodiments, the method for treatingvulvar and/or vaginal itching or irritation includes reducing theitching/irritation severity score from 2, prior to treatment of asubject, to 1, after 2 weeks of treatment of the subject. In someembodiments, the method for treating vulvar and/or vaginal itching orirritation includes reducing the itching/irritation severity score from1, prior to treatment of subject, to 0, after 2 weeks of treatment ofthe subject.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes reducing the itching/irritation severityscore from 3, prior to treatment of a subject, to 2, after 6 weeks oftreatment of the subject. In some embodiments, the method for treatingvulvar and/or vaginal itching or irritation includes reducing theitching/irritation severity score from 2, prior to treatment of asubject, to 1, after 6 weeks of treatment of the subject. In someembodiments, the method for treating vulvar and/or vaginal itching orirritation includes reducing the itching/irritation severity score from1, prior to treatment of subject, to 0, after 6 weeks of treatment ofthe subject.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes reducing the itching/irritation severityscore from 3, prior to treatment of a subject, to 2, after 8 weeks oftreatment of the subject. In some embodiments, the method for treatingvulvar and/or vaginal itching or irritation includes reducing theitching/irritation severity score from 2, prior to treatment of asubject, to 1, after 8 weeks of treatment of the subject. In someembodiments, the method for treating vulvar and/or vaginal itching orirritation includes reducing the itching/irritation severity score from1, prior to treatment of subject, to 0, after 8 weeks of treatment ofthe subject.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes reducing the itching/irritation severityscore from 3, prior to treatment of a subject, to 2, after 12 weeks oftreatment of the subject. In some embodiments, the method for treatingvulvar and/or vaginal itching or irritation includes reducing theitching/irritation severity score from 2, prior to treatment of asubject, to 1, after 12 weeks of treatment of the subject. In someembodiments, the method for treating vulvar and/or vaginal itching orirritation includes reducing the itching/irritation severity score from1, prior to treatment of subject, to 0, after 12 weeks of treatment ofthe subject.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes decreasing the severity ofitching/irritation after two weeks of treatment, wherein the severity isassessed on a scale of 0-3 points, and the average decrease ranges froma 0.3-point decrease to a 0.6-point decrease. The average decrease canbe determined by observing any suitable number of subjects. In someembodiments, the number of subjects is at least 100. In someembodiments, the number of subjects is at least 500. In someembodiments, the number of subjects ranges from 700 to 800. In someembodiments, the number of subjects ranges from 740 to 750. In someembodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes decreasing the severity ofitching/irritation after six weeks of treatment, wherein the severity isassessed on a scale of 0-3 points, and the average decrease ranges froma 0.5-point decrease to a 0.7-point decrease. The average decrease canbe determined by observing any suitable number of subjects. In someembodiments, the number of subjects is at least 100. In someembodiments, the number of subjects is at least 500. In someembodiments, the number of subjects ranges from 700 to 800. In someembodiments, the number of subjects ranges from 740 to 750. In someembodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes decreasing the severity ofitching/irritation after eight weeks of treatment, wherein the severityis assessed on a scale of 0-3 points, and the average decrease rangesfrom a 0.5-point decrease to a 0.8-point decrease. The average decreasecan be determined by observing any suitable number of subjects. In someembodiments, the number of subjects is at least 100. In someembodiments, the number of subjects is at least 500. In someembodiments, the number of subjects ranges from 700 to 800. In someembodiments, the number of subjects ranges from 740 to 750. In someembodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes decreasing the severity ofitching/irritation after twelve weeks of treatment, wherein the severityis assessed on a scale of 0-3 points, and the average decrease rangesfrom a 0.5-point decrease to a 1.0-point decrease. The average decreasecan be determined by observing any suitable number of subjects. In someembodiments, the number of subjects is at least 100. In someembodiments, the number of subjects is at least 500. In someembodiments, the number of subjects ranges from 700 to 800. In someembodiments, the number of subjects ranges from 740 to 750. In someembodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating vulvar and/or vaginalitching or irritation includes administering a suppository so as toprovide an estradiol C_(max) or AUC as described herein. According toembodiments, a method for treating vulvar and/or vaginal itching orirritation is provided, the method including administering a suppositoryso as to provide an estrone C_(max) or AUC as described herein.

According to embodiments, a method for treating dyspareunia is provided.The method includes administration of a suppository or dosage asdescribed herein. Treating dyspareunia according to the method disclosedherein can include, decreasing the severity of dyspareunia by 1%, 5%,10%, 15%, 20%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99%. The decrease in severity can be obtained following 2weeks of treatment, or 6 weeks of treatment, or 8 weeks of treatment, or12 weeks of treatment. In some embodiments, dyspareunia is assessedusing a severity scale, ranging from 0 to 4 points wherein 0 indicatesno pain associated with sexual activity (with vaginal penetration), 1indicates mild pain associated with sexual activity (with vaginalpenetration), 2 indicates moderate pain associated with sexual activity(with vaginal penetration), and 3 indicates severe pain associated withsexual activity (with vaginal penetration).

In some embodiments, the method for treating dyspareunia includesreducing the dyspareunia severity score from 3, prior to treatment of asubject, to 2, after 2 weeks of treatment of the subject. In someembodiments, the method for treating dyspareunia includes reducing thedyspareunia severity score from 2, prior to treatment of a subject, to1, after 2 weeks of treatment of the subject. In some embodiments, themethod for treating dyspareunia includes reducing the dyspareuniaseverity score from 1, prior to treatment of subject, to 0, after 2weeks of treatment of the subject.

In some embodiments, the method for treating dyspareunia includesreducing the dyspareunia severity score from 3, prior to treatment of asubject, to 2, after 6 weeks of treatment of the subject. In someembodiments, the method for treating dyspareunia includes reducing thedyspareunia severity score from 2, prior to treatment of a subject, to1, after 6 weeks of treatment of the subject. In some embodiments, themethod for treating dyspareunia includes reducing the dyspareuniaseverity score from 1, prior to treatment of subject, to 0, after 6weeks of treatment of the subject.

In some embodiments, the method for treating dyspareunia includesreducing the dyspareunia severity score from 3, prior to treatment of asubject, to 2, after 8 weeks of treatment of the subject. In someembodiments, the method for treating dyspareunia includes reducing thedyspareunia severity score from 2, prior to treatment of a subject, to1, after 8 weeks of treatment of the subject. In some embodiments, themethod for treating dyspareunia includes reducing the dyspareuniaseverity score from 1, prior to treatment of subject, to 0, after 8weeks of treatment of the subject.

In some embodiments, the method for treating dyspareunia includesreducing the dyspareunia severity score from 3, prior to treatment of asubject, to 2, after 12 weeks of treatment of the subject. In someembodiments, the method for treating dyspareunia includes reducing thedyspareunia severity score from 2, prior to treatment of a subject, to1, after 12 weeks of treatment of the subject. In some embodiments, themethod for treating dyspareunia includes reducing the dyspareuniaseverity score from 1, prior to treatment of subject, to 0, after 12weeks of treatment of the subject.

In some embodiments, the method for treating dyspareunia includesdecreasing the severity of dyspareunia after two weeks of treatment,wherein the severity is assessed on a scale of 0-3 points, and theaverage decrease ranges from a 0.9-point decrease to a 1.1-pointdecrease. The average decrease can be determined by observing anysuitable number of subjects. In some embodiments, the number of subjectsis at least 100. In some embodiments, the number of subjects is at least500. In some embodiments, the number of subjects ranges from 700 to 800.In some embodiments, the number of subjects ranges from 740 to 750. Insome embodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating dyspareunia includesdecreasing the severity of dyspareunia after six weeks of treatment,wherein the severity is assessed on a scale of 0-3 points, and theaverage decrease ranges from a 1.3-point decrease to a 1.5-pointdecrease. The average decrease can be determined by observing anysuitable number of subjects. In some embodiments, the number of subjectsis at least 100. In some embodiments, the number of subjects is at least500. In some embodiments, the number of subjects ranges from 700 to 800.In some embodiments, the number of subjects ranges from 740 to 750. Insome embodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating dyspareunia includesdecreasing the severity of dyspareunia after eight weeks of treatment,wherein the severity is assessed on a scale of 0-3 points, and theaverage decrease ranges from a 1.5-point decrease to a 1.8-pointdecrease. The average decrease can be determined by observing anysuitable number of subjects. In some embodiments, the number of subjectsis at least 100. In some embodiments, the number of subjects is at least500. In some embodiments, the number of subjects ranges from 700 to 800.In some embodiments, the number of subjects ranges from 740 to 750. Insome embodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating dyspareunia includesdecreasing the severity of dyspareunia after twelve weeks of treatment,wherein the severity is assessed on a scale of 0-3 points, and theaverage decrease ranges from a 1.5-point decrease to a 1.8-pointdecrease. The average decrease can be determined by observing anysuitable number of subjects. In some embodiments, the number of subjectsis at least 100. In some embodiments, the number of subjects is at least500. In some embodiments, the number of subjects ranges from 700 to 800.In some embodiments, the number of subjects ranges from 740 to 750. Insome embodiments, the vaginal estradiol formulation contains 4 μg ofestradiol. In some embodiments, the vaginal estradiol formulationcontains 10 μg of estradiol. In some embodiments, the vaginal estradiolformulation contains 25 μg of estradiol.

In some embodiments, the method for treating dyspareunia includesadministering a suppository so as to provide an estradiol C_(max) or AUCas described herein. According to embodiments, a method for treatingdyspareunia is provided, the method including administering asuppository so as to provide an estrone C_(max) or AUC as describedherein.

According to embodiments, a method for treating urinary tract infectionsis provided. As used herein the term “urinary tract infection” refers toan infection of the kidneys, ureters, bladder and urethra by amicroorganism such as Escherichia coli, Staphylococcus saprophyticus,Klebsiella sp., Enterobacter sp., or Proteus sp. The method for treatingurinary tract infections generally includes administering a soft gelvaginal estradiol formulation (i.e., a suppository) as described herein.According to certain embodiments, the method further includes decreasingurethral discomfort, frequency or urination, hematuria, dysuria, and/orstress incontinence. According to certain embodiments, a method fortreating urinary tract infections is provided, the method includingadministering a suppository as described herein and decreasing vaginalpH from above 4.5 to between 3.5 and 4.5 (inclusive). The method can beparticularly effective for treating urinary tract infections in elderlysubjects (e.g., subjects older than 65 years, or older than 75 years, orolder than 85 years). According to embodiments, a method for treatingurinary tract infections is provided, the method including administeringa suppository so as to provide an estradiol C_(max) or AUC as describedherein. According to embodiments, a method for treating urinary tractinfections is provided, the method including administering a suppositoryso as to provide an estrone C_(max) or AUC as described herein.According to embodiments, a method for treating sexual dysfunction isprovided. As used herein with respect to female subjects, the term“sexual dysfunction” generally refers to pain or discomfort duringsexual intercourse, diminished vaginal lubrication, delayed vaginalengorgement, increased time for arousal, diminished ability to reachorgasm, diminished clitoral sensation, diminished sexual desire, and/ordiminished arousal. According to embodiments, a method for treatingsexual dysfunction is provided, the method including administering asuppository so as to provide an estradiol C_(max) or AUC as describedherein. According to embodiments, a method for treating sexualdysfunction is provided, the method including administering asuppository so as to provide an estrone C_(max) or AUC as describedherein.

Sexual function and dysfunction can be assessed using the Female SexualFunction Index (FSFI) (see, Rosen R, Brown C, Heiman J, et al. “TheFemale Sexual Function Index (FSFI): A Multidimensional Self-ReportInstrument for the Assessment of Female Sexual Function.” Journal of Sex& Marital Therapy 2000. 26: p. 191-208). The FSFI is useful forassessing various domains of sexual functioning (e.g. sexual desire,arousal, orgasm, satisfaction and pain). Accordingly, the method fortreating sexual dysfunction as provided herein can include administeringa vaginal soft gel formulation to a subject and increasing a subject'sfull-scale FSFI score, FSFI-desire score, FSFI-arousal score,FSFI-lubrication score and/or FSFI-orgasm score.

Female Sexual Function Index (FSFI)

Question Answer Options Q1: Over the past 4 weeks, how often 5 = Almostalways or always did you feel sexual desire or interest? 4 = Most times(more than half the time) 3 = Sometimes (about half the time) 2 = A fewtimes (less than half the time) 1 = Almost never or never Q2: Over thepast 4 weeks, how would you 5 = Very high rate your level (degree) ofsexual desire 4 = High or interest? 3 = Moderate 2 = Low 1 = Very low ornone at all Q3. Over the past 4 weeks, how often did 0 = No sexualactivity you feel sexually aroused (“turned on”) 5 = Almost always oralways during sexual activity or intercourse? 4 = Most times (more thanhalf the time) 3 = Sometimes (about half the time) 2 = A few times (lessthan half the time) 1 = Almost never or never Q4. Over the past 4 weeks,how would you 0 = No sexual activity rate your level of sexual arousal(“turn 5 = Very high on”) during sexual activity or intercourse? 4 =High 3 = Moderate 2 = Low 1 = Very low or none at all Q5. Over the past4 weeks, how confident 0 = No sexual activity were you about becomingsexually aroused 5 = Very high confidence during sexual activity orintercourse? 4 = High confidence 3 = Moderate confidence 2 = Lowconfidence 1 = Very low or no confidence Q6. Over the past 4 weeks, howoften have 0 = No sexual activity you been satisfied with your arousal 5= Almost always or always (excitement) during sexual activity or 4 =Most times (more than half the time) intercourse? Response Options 3 =Sometimes (about half the time) 2 = A few times (less than half thetime) 1 = Almost never or never Q7: Over the past 4 weeks, how often did0 = No sexual activity you become lubricated (“wet”) during 5 = Almostalways or always sexual activity or intercourse? 4 = Most times (morethan half the time) 3 = Sometimes (about half the time) 2 = A few times(less than half the time) 1 = Almost never or never Q8. Over the past 4weeks, how difficult 0 = No sexual activity was it to become lubricated(“wet”) during 1 = Extremely difficult or impossible sexual activity orintercourse? 2 = Very difficult 3 = Difficult 4 = Slightly difficult 5 =Not difficult Q9: Over the past 4 weeks, how often did 0 = No sexualactivity you maintain your lubrication (“wetness”) 5 = Almost always oralways until completion of sexual activity 4 = Most times (more thanhalf the time) or intercourse? 3 = Sometimes (about half the time) 2 = Afew times (less than half the time) 1 = Almost never or never Q10: Overthe past 4 weeks, how difficult 0 = No sexual activity was it tomaintain your lubrication 1 = Extremely difficult or impossible(“wetness”) until completion of sexual 2 = Very difficult activity orintercourse? 3 = Difficult 4 = Slightly difficult 5 = Not difficult Q11.Over the past 4 weeks, when you had 0 = No sexual activity sexualstimulation or intercourse, how 5 = Almost always or always often didyou reach orgasm (climax)? 4 = Most times (more than half the time) 3 =Sometimes (about half the time) 2 = A few times (less than half thetime) 1 = Almost never or never Q12: Over the past 4 weeks, when you had0 = No sexual activity sexual stimulation or intercourse, how 1 =Extremely difficult or impossible difficult was it for you to reachorgasm 2 = Very difficult (climax)? 3 = Difficult 4 = Slightly difficult5 = Not difficult Q13: Over the past 4 weeks, how satisfied 0 = Nosexual activity were you with your ability to reach orgasm 5 = Verysatisfied 4 (climax) during sexual activity or 4 = Moderately satisfiedintercourse? 3 = About equally satisfied and dissatisfied 2 = Moderatelydissatisfied 1 = Very dissatisfied Q14: Over the past 4 weeks, howsatisfied 0 = No sexual activity have you been with the amount ofemotional 5 = Very satisfied closeness during sexual activity 4 =Moderately satisfied between you and your partner? 3 = About equallysatisfied and dissatisfied 2 = Moderately dissatisfied 1 = Verydissatisfied Q15: Over the past 4 weeks, how satisfied 5 = Verysatisfied have you been with your sexual relationship 4 = Moderatelysatisfied with your partner? 3 = About equally satisfied anddissatisfied 2 = Moderately dissatisfied 1 = Very dissatisfied Q16: Overthe past 4 weeks, how satisfied 5 = Very satisfied have you been withyour overall sexual life? 4 = Moderately satisfied 3 = About equallysatisfied and dissatisfied 2 = Moderately dissatisfied 1 = Verydissatisfied Q17: Over the past 4 weeks, how often did 0 = Did notattempt intercourse you experience discomfort or pain during 1 = Almostalways or always vaginal penetration? 2 = Most times (more than half thetime) 3 = Sometimes (about half the time) 4 = A few times (less thanhalf the time) 5 = Almost never or never Q18: Over the past 4 weeks, howoften did 0 = Did not attempt intercourse you experience discomfort orpain following 1 = Almost always or always vaginal penetration? 2 = Mosttimes (more than half the time) 3 = Sometimes (about half the time) 4 =A few times (less than half the time) 5 = Almost never or never Q19.Over the past 4 weeks, how would you 0 = Did not attempt intercourserate your level (degree) of discomfort or pain 1 = Very high during orfollowing vaginal penetration? 2 = High 3 = Moderate 4 = Low 5 = Verylow or none at all

FSFI Scoring System

Domain Questions Score Range Factor Minimum Maximum Desire 1, 2 1-5 0.61.2 6.0 Arousal 3, 4, 5, 6 0-5 0.3 0 6.0 Lubrication 7, 8, 9, 10 0-5 0.30 6.0 Orgasm 11, 12, 13 0-5 0.4 0 6.0 Satisfaction 14, 15, 16 0 (or1)-5     0.4 0.8 6.0 Pain 17, 18, 19 0-5 0.4 0 6.0 Full Scale ScoreRange: 2.0 36.0

In some embodiments, the method for treating sexual dysfunction includesadministering estradiol to the subject and increasing the FSFI-desirescore by at least about 20%, or at least about 25%, or at least about30% as compared to baseline.

In some embodiments, the method for treating sexual dysfunction includesadministering estradiol to the subject and increasing the FSFI-arousalscore by at least about 30%, or at least about 40%, or at least about50% as compared to baseline.

In some embodiments, the method for treating sexual dysfunction includesadministering estradiol to the subject and increasing theFSFI-lubrication score by at least about 85%, or at least about 95%, orat least about 115% as compared to baseline.

In some embodiments, the method for treating sexual dysfunction includesadministering estradiol to the subject and increasing the FSFI-orgasmscore by at least about 40%, or at least about 60% as compared tobaseline.

In some embodiments, the method for treating sexual dysfunction includesadministering estradiol to the subject and increasing the total FSFIscore by at least about 50%, or at least about 55%, or at least about70% as compared to baseline.

Examples of other metrics for assessment of sexual function include, butare not limited to, Changes in Sexual Function Questionnaire (“CSFQ”;Clayton et al., Psychopharmacol Bull. 33(4):731-45 (1997) and Clayton etal., Psychopharmacol. Bull. 33(4):747-53 (1997)); the DerogatisInterview for Sexual Functioning—Self-Report (“DISF-SR”; Derogatis, JSex Marital Ther. 23:291-304 (1997)); the Golombok-Rust Inventory ofSexual Satisfaction (“GRISS”; Rust et al., Arch. Sex Behav. 15:157-165(1986)); the Sexual Function Questionnaire (“SFQ”; Quirk et al., JWomens Health Gend Based Med. 11:277-289 (2002)); and the Arizona SexualExperience Scale (“ASEX”; McGahuey et al., J Sex Marital Ther. 26:25-40(2000)), the entire disclosures of which are incorporated herein byreference. For assessment using a questionnaire, a measure of sexualdysfunction function is increased when the score in the appropriatedomain, subscale or subtest is indicative of sexual dysfunction, asestablished for that questionnaire. For instance, a female's sexualinterest is considered reduced, when assessed using the CSFQ, if thesubscale for sexual interest score is less than or equal to 9.Conversely, sexual dysfunction is considered improved when the score inthe appropriate domain, subscale or subtest is indicative of higher(e.g., normal or desired) sexual function. For a clinician's assessment,sexual dysfunction may be assessed in comparison to a previous point intime for the patient and/or in comparison to a patient's peers withrespect to age, gender, sexual experience, and health, or may also bedetermined via a validated questionnaire administered by the clinician.

According to embodiments, the efficacy and safety of the pharmaceuticalcompositions described herein in the treatment of the symptoms of VVAmay be determined. According to embodiments, the size, effect, cytology,histology, and variability of the VVA may be determined using variousendpoints to determine efficacy and safety of the pharmaceuticalcompositions described herein or as otherwise accepted in the art, atpresent or as further developed. One source of endpoints is with the USFood and Drug Administration's (FDA) published guidelines for treatmentof VVA with estradiol.

According to embodiments, a method of treating VVA, includingdyspareunia, vaginal dryness, and estrogen-deficient urinary states(including urinary tract infections), is provided that allows a subjectto be ambulatory immediately or within minutes after a gelatin capsulecontaining the pharmaceutical compositions disclosed herein areadministered. According to embodiments, a gelatin capsule containing apharmaceutical composition as disclosed herein is administered bydigitally inserting the gelatin capsule containing the pharmaceuticalcomposition into the vagina approximately two inches or inserting intothe third of the vagina closest to the vaginal opening as shown in FIGS.26A, 26B, and 26C. According to embodiments, the gelatin capsule adheresto the vaginal tissue and dissolves, ruptures, or otherwisedisintegrates soon after being inserted into the vagina therebyreleasing the pharmaceutical composition. The pharmaceutical compositionspreads onto the vaginal tissue and is rapidly absorbed. According toembodiments, the gelatin capsule is also fully absorbed by the vaginaltissue. According to some embodiments, a viscosity enchancer such asTEFOSE 63 provides increased viscosity to ensure the pharmaceuticalcomposition stays within the desired absorption area, therebyestrogenizing the vagina, labia, and/or vulva. The combination of highviscosity, bioadhesion, and rapid absorption prevents the need forsubjects to remain supine after administration to allow the tissue toabsorb the estradiol, thereby allowing subjects to be ambulatoryimmediately or almost immediately after administration.

According to embodiments, a method for treating VVA, includingdyspareunia, vaginal dryness, and estrogen-deficient urinary states(including urinary tract infections), without causing non-naturaldischarge (e.g., discharge of a pharmaceutical composition or acomponent thereof) is provided. According to the method, a soft gelatincapsule is administered containing a liquid pharmaceutical compositionthat is able to be fully absorbed by the vaginal tissue. According toembodiments, the pharmaceutical composition itself is fully absorbed bythe vaginal tissue. According to embodiments, the pharmaceuticalcomposition and gelatin capsule are administered in a volume and size,respectively, that allows a subject's vaginal tissue to fully absorb thepharmaceutical composition. According to embodiments, such absorptionwill occur contemporaneously with the subject being ambulatory.According to the method, the gelatin capsule and liquid pharmaceuticalcomposition are fully absorved by the vaginal tissue, wherein the onlydischarge that occurs after estrogenizing the vagina is naturaldischarge that a woman would have experienced prior to menopause.“Natural” vaginal discharge refers to a small amount of fluid that flowsout of the vagina each day, carrying out old cells that have lined thevagina. Natural discharge is usually clear or milky. Non-naturaldischarge can refer to discharge that is higher in volume than naturaldischarge, different in color than natural discharge, or different inconsistency than natural discharge. Non-natural discharge can also referto the discharge (e.g., leaking) of a pharmaceutical composition fromthe vagina.

According to embodiments, a method of treating VVA, includingdyspareunia, vaginal dryness, and estrogen-deficient urinary states(including urinary tract infections), using a liquid pharmaceuticalcomposition is provided. According to the method, a soft gelatin capsulecontaining a liquid composition for treating VVA is provided to asubject. The subject inserts the soft gelatin capsule containing theliquid composition for treating VVA into their vagina either digitallyor with an applicator, wherein the soft gelatin capsule dissolves,ruptures, or disintegrates and the liquid composition is released intothe vagina. According to embodiments, the liquid composition fortreating VVA is a pharmaceutical composition disclosed herein. Accordingto embodiments, the subject inserts the gelatin capsule about two inchesinto the vagina, or in the third of the vagina closest to the vaginalopening. According to embodiments, the subject is ambulatory immediatelyafter or soon after administration.

According to embodiments, a method is disclosed herein for treating VVA,including dyspareunia, vaginal dryness, and estrogen-deficient urinarystates (including urinary tract infections), comprising improving thesymptoms of VVA, compared to placebo or baseline, within two weeks byvaginally administering a composition for the treatment of VVA. One ofskill in the art will understand that the improvements can be assessedstatistically as described herein, and that any improvement can be astatistically significant improvement. According to embodiments, thecomposition for the treatment of VVA is a liquid pharmaceuticalcomposition as disclosed herein. According to embodiments, thecomposition for the treatment of VVA is a liquid containing from 1 μg to25 μg of estradiol. According to embodiments, the method ofadministration is a method disclosed herein, including the insertionmethod shown in FIGS. 26A, 26B, and 26C. According to embodiments, atthe two week point of measurement, the estradiol is not detectedsystemically when measured using standard pharmaceutical pharmacokineticparameters, such as AUC and C_(max).

According to embodiments, a method is disclosed herein for treating VVA,including dyspareunia, vaginal dryness, and estrogen-deficient urinarystates (including urinary tract infections), comprising improving thesymptoms of VVA, compared to placebo or baseline, within four weeks byvaginally administering a composition for the treatment of VVA. One ofskill in the art will understand that the improvements can be assessedstatistically as described herein, and that any improvement can be astatistically significant improvement. According to embodiments, thecomposition for the treatment of VVA is a liquid pharmaceuticalcomposition as disclosed herein. According to embodiments, thecomposition for the treatment of VVA is a liquid containing from 1 μg to25 μg of estradiol. According to embodiments, the method ofadministration is a method disclosed herein, including the insertionmethod shown in FIGS. 26A, 26B, and 26C. According to embodiments, atthe two week point of measurement and/or the four week point ofmeasurement, the estradiol is not detected systemically when measuredusing standard pharmaceutical pharmacokinetic parameters, such as AUCand C_(max).

According to embodiments, a method is disclosed herein for treating VVA,including dyspareunia, vaginal dryness, and estrogen-deficient urinarystates (including urinary tract infections), comprising improving thesymptoms of VVA, compared to placebo or baseline, within eight weeks byvaginally administering a composition for the treatment of VVA. One ofskill in the art will understand that the improvements can be assessedstatistically as described herein, and that any improvement can be astatistically significant improvement. According to embodiments, thecomposition for the treatment of VVA is a liquid pharmaceuticalcomposition as disclosed herein. According to embodiments, thecomposition for the treatment of VVA is a liquid containing from 1 μg to25 μg of estradiol. According to embodiments, the method ofadministration is a method disclosed herein, including the insertionmethod shown in FIGS. 26A, 26B, and 26C. According to embodiments, atthe two week point of measurement and/or the eight week point ofmeasurement, the estradiol is not detected systemically when measuredusing standard pharmaceutical pharmacokinetic parameters, such as AUCand C_(max).

According to embodiments, a method is disclosed herein for treating VVA,including dyspareunia, vaginal dryness, and estrogen-deficient urinarystates (including urinary tract infections), comprising improving thesymptoms of VVA, compared to placebo or baseline, within ten weeks byvaginally administering a composition for the treatment of VVA. One ofskill in the art will understand that the improvements can be assessedstatistically as described herein, and that any improvement can be astatistically significant improvement. According to embodiments, thecomposition for the treatment of VVA is a liquid pharmaceuticalcomposition as disclosed herein. According to embodiments, thecomposition for the treatment of VVA is a liquid containing from 1 μg to25 μg of estradiol. According to embodiments, the method ofadministration is a method disclosed herein, including the insertionmethod shown in FIGS. 26A, 26B, and 26C. According to embodiments, atthe two week point of measurement and/or the ten week point ofmeasurement, the estradiol is not detected systemically when measuredusing standard pharmaceutical pharmacokinetic parameters, such as AUCand C_(max).

According to embodiments, a method for treating VVA, includingdyspareunia, vaginal dryness, and estrogen-deficient urinary states(including urinary tract infections), comprising administering acomposition containing estradiol for the treatment of VVA is provided,wherein the method improves the symptoms of VVA, compared with baselineor placebo, in at least one of two weeks, four weeks, six weeks, eightweeks, or twelve weeks, wherein the estradiol is not detectedsystemically using standard pharmaceutical pharmacokinetic parameters,such as AUC and C_(max). One of skill in the art will understand thatthe improvements can be assessed statistically as described herein, andthat any improvement can be a statistically significant improvement.According to embodiments, the composition containing estradiol is aliquid composition as disclosed herein. According to embodiments, thecopomosition contains 1 μg to 25 μg of estradiol.

According to embodiments, a method for reestrogenizing the vagina,labia, or vulva is provided, wherein the method comprises administeringa composition containing estradiol for the treatment of VVA, wherein thecomposition is a liquid containing estradiol or a synthetic estrogen,and wherein the liquid spreads over a surface area of the vagina, labia,or vulva which is larger than the area covered by a solid composition.For example, the liquid can spread over a surface area ranging fromabout 50 cm² to about 120 cm² (e.g., from about 50 cm² to about 60 cm²;or from about 60 cm² to about 70 cm²; or from about 70 cm² to about 80cm²; or from about 80 cm² to about 90 cm²; or from about 90 cm² to about100 cm²; or from about 100 cm² to about 110 cm²; or from about 110 cm²to about 120 cm²; or from about 65 cm² to about 110 cm²). According toembodiments, the subject inserts a liquid composition into her vagina ina capsule, such as a hard or soft gelatin capsule, that then dissolves,ruptures, disintegrates, or otherwise releases the liquid in the vagina.According to embodiments, the liquid contains at least one of abio-adhesive or viscosity enhancer to prevent the liquid fromdischarging from the vagina before the estradiol or synthetic estrogencan be absorbed into the vaginal tissue in a dose sufficient to effectreestrongenization of the vagina. According to embodiments, the vaginawill be statistically significantly reestrogenized within two weeks ofadministration compared to baseline or placebo levels. According toembodiments, the vagina will be statistically significantlyreestrogenized within four weeks of administration compared to baselineor placebo levels.

According to embodiments, the vagina will be statistically significantlyreestrogenized within six weeks of administration compared to baselineor placebo levels. According to embodiments, the vagina will bestatistically significantly reestrogenized within eight weeks ofadministration compared to baseline or placebo levels. According toembodiments, the vagina will be statistically significantlyreestrogenized within ten weeks of administration compared to baselineor placebo levels. According to embodiments, the vagina will bestatistically significantly reestrogenized within twelve or more weeksof administration compared to baseline or placebo levels.

VII. MEASUREMENT OF EFFICACY

According to embodiments, administration of the pharmaceuticalcompositions described herein resulted in treatment of the VVA, as wellas improvement of one or more of the associated symptoms. Patients withVVA experience shrinking of the vaginal canal in both length anddiameter and the vaginal canal has fewer glycogen-rich vaginal cells tomaintain moisture and suppleness. In addition, the vaginal wall canbecome thin, pale, dry, or sometimes inflamed (atrophic vaginitis).These changes can manifest as a variety of symptoms collectivelyreferred to as VVA. Such symptoms include, without limitations, anincrease in vaginal pH; reduction of vaginal epithelial integrity,vaginal secretions, or epithelial surface thickness; pruritus; vaginaldryness; dyspareunia (pain or bleeding during sexual intercourse);urinary tract infections; or a change in vaginal color. According toembodiments, efficacy is measured as a reduction of vulvar and vaginalatrophy in a patient back to premenopausal conditions. According toembodiments, the change is measured as a reduction in the severity ofone or more atrophic effects measured at baseline (screening, Day 1) andcompared to a measurement taken at Day 15 (end of treatment). Severityof the atrophic effect may be measured using a scale of 0 to 3 where,for example, none=0, mild=1, moderate=2, or severe=3. Such scoring isimplemented to evaluate the pre-treatment condition of patients; todetermine the appropriate course of a treatment regime; such as dosage,dosing frequency, and duration, among others; and post-treatmentoutcomes.

One of the symptoms of VVA is increased vaginal pH. In further aspectsof this disclosure, treatment with the pharmaceutical compositionsdescribed herein resulted in a decrease in vaginal pH. A decrease invaginal pH is measured as a decrease from the vaginal pH at baseline(screening) to the vaginal pH at Day 15, according to embodiments. Insome embodiments, a pH of 5 or greater may be associated with VVA. Insome embodiments, pH is measured using a pH indicator strip placedagainst the vaginal wall. In some embodiments, a change in vaginal pH isa change in a patient's vaginal pH to a pH of less than about pH 5.0. Insome embodiments, a subject's vaginal pH may be less than about pH 4.9,pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1, pH 4.0,pH 3.9, pH 3.8, pH 3.7, pH 3.6, or pH 3.5.

According to embodiments, treatment with the pharmaceutical compositionsdescribed herein resulted in improvements in the vaginal MaturationIndex. The Maturation Index is measured as a change in cell composition.According to embodiments and as related to VVA, a change in cellcomposition is measured as the change in percent of composition oramount of parabasal vaginal cells, intermediate cells, and superficialvaginal cells, such as a change in the composition or amount ofparabasal vaginal cells compared with or, relative to, a change insuperficial vaginal cells. A subject having VVA symptoms often has anincreased number of parabasal cells and a reduced number of superficialcells (e.g., less than about 5%) compared with women who do not sufferfrom VVA. Conversely, a subject having decreasing VVA symptoms, or asotherwise responding to treatment, may demonstrate an improvement in theMaturation Index, specifically a decrease in the amount of parabasalcells or an increase in the amount of superficial cells compared tobaseline (screening). In embodiments, a decrease in parabasal cells ismeasured as a reduction in the percent of parabasal cells; the percentreduction may be at least about an 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% reduction in the number ofparabasal cells. In embodiments, a percent reduction may be at leastabout a 54% reduction in the number of parabasal cells. In embodiments,an increase in superficial cells is measured as an increase in thepercent of superficial cells; the percent increase in superficial cellsmay be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%increase in the number of superficial cells. In further embodiments, apercent increase may be at least about a 35% increase in the number ofsuperficial cells.

In some embodiments, an improvement in the Maturation Index is assessedas a change over time. For example, as a change in cell compositionmeasured at a baseline (screening) at Day 1 compared to the cellcomposition measured at Day 15. The change in cell composition may alsobe assessed as a change in the amount of parabasal cells over time,optionally in addition to measuring changes in parabasal cells andsuperficial cells as described above. Such cells may be obtained fromthe vaginal mucosal epithelium through routine gynecological examinationand examined by means of a vaginal smear.

In various further aspects of this disclosure, treatment with thepharmaceutical compositions described herein resulted in any of: anincrease in superficial cells; a decrease in parabasal cells; and anincrease in intermediate cells.

In further aspects of this disclosure, samples may be collected todetermine hormone levels, in particular, estradiol levels. In someembodiments, blood samples may be taken from a subject and the level ofestradiol measured (pg/mL). In some embodiments, estradiol levels may bemeasured at 0 hours (for example, at time of first treatment), at 1 hour(for example, post first treatment), at 3 hours, and at 6 hours. In someembodiments, samples may be taken at day 8 (for example, post firsttreatment) and at day 15 (for example, one day post the last treatmenton day 14). In some embodiments, descriptive statistics of plasmaestradiol concentrations at each sampling time and observed C_(max) andT_(max) values may be measured and the AUC calculated.

In some embodiments, a suppository can comprise about 25 μg ofestradiol. In such cases, administration of the suppository to a patientcan provide, in a plasma sample from the patient, parameters includingone or more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estradiol of about 19 pg*hr/mL toabout 29 pg*hr/mL (e.g., 19.55 pg*hr/mL to about 28.75 pg*hr/mL); or 2)a corrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiolof about 75 pg*hr/mL to about 112 pg*hr/mL (e.g., 75.82 pg*hr/mL toabout 111.50). In some embodiments, administration of the suppository toa patient provides, in a plasma sample from the patient, one or moreparameters selected from: 1) a corrected geometric mean peak plasmaconcentration (C_(max)) of estrone of about 9 pg*hr/mL to about 14pg*hr/mL (e.g., 9.17 pg*hr/mL to about 13.49 pg*hr/mL); and 2) acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estrone ofabout 43 pg*hr/mL to about 65 pg*hr/mL (e.g., 43.56 pg*hr/mL to about64.06 pg*hr/mL). In some embodiments, administration of the suppositoryto a patient provides, in a plasma sample from the patient, provides oneor more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 416 pg*hr/mLto about 613 pg*hr/mL (e.g., 416.53 pg*hr/mL to about 612.55 pg*hr/mL);and 2) a corrected geometric mean area under the curve (AUC)₀₋₂₄ ofestrone sulfate of about 3598 pg*hr/mL to about 5291 pg*hr/mL (e.g.,3598.04 pg*hr/mL to about 5291.24 pg*hr/mL).

In some embodiments, a suppository includes about 25 μg of estradiol. Insome such embodiments, administration of the suppository to a patientcan provide, in a plasma sample from the patient, parameters includingone or more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estradiol ranging from about 20.9pg/mL to about 32.8 pg/mL (e.g., 20.96 pg/mL to about 32.75 pg/mL); 2) acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiolranging from about 104.3 pg*hr/mL to about 163.1 pg*hr/mL (e.g., 104.32pg*hr/mL to about 163.0 pg*hr/mL); and 3) an average concentration(C_(avg)) of estradiol ranging from about 4.3 pg/mL to about 6.8 pg/mL(e.g., 4.32 pg/mL to about 6.75 pg/mL), as assessed at day 1.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient can provide, in a plasma sample from thepatient, parameters including one or more parameters selected from: 1) acorrected geometric mean peak plasma concentration (C_(max)) ofestradiol of about 26.2 pg/mL; 2) a corrected geometric mean area underthe curve (AUC)₀₋₂₄ of estradiol of about 130 pg*hr/mL; and 3) anaverage concentration (C_(avg)) of estradiol of about 5.4 pg/mL, asassessed at day 1.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient can provide, in a plasma sample from thepatient, parameters including one or more parameters selected from: 1) acorrected geometric mean peak plasma concentration (C_(max)) ofestradiol ranging from about 9.5 pg/mL to about 15.1 pg/mL (e.g., 9.60pg*hr/mL to about 15.00 pg/mL); 2) a corrected geometric mean area underthe curve (AUC)₀₋₂₄ of estradiol ranging from about 67.6 pg*hr/mL toabout 105.8 pg*hr/mL (e.g., 67.68 pg*hr/mL to about 105.75 pg*hr/mL);and 3) an average concentration (C_(avg)) of estradiol ranging fromabout 2.7 pg/mL to about 4.4 pg/mL (e.g., 2.80 pg/mL to about 4.38pg/mL) of estradiol as assessed at day 14.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient can provide, in a plasma sample from thepatient, parameters including one or more parameters selected from: 1) acorrected geometric mean peak plasma concentration (C_(max)) ofestradiol of about 12.0 pg/mL; 2) a corrected geometric mean area underthe curve (AUC)₀₋₂₄ of estradiol of about 84.6 pg*hr/mL; and 3) anaverage concentration (C_(avg)) of estradiol of about 3.5 pg/mL, asassessed at day 14.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone conjugates rangingfrom about 158.8 pg/mL to about 248.3 pg/mL (e.g., 158.88 hr/mL to about248.25 pg*hr/mL); and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone conjugates ranging from about 1963.1 pg*hr/mL toabout 3067.6 pg*hr/mL (e.g., 1963.20 pg*hr/mL to about 3067.50 pg*hr/mL)as assessed at day 1.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone conjugates of about198.6 pg/mL; and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone conjugates of about 2454 pg*hr/mL as assessed atday 1.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estradiol ranging fromabout 173.5 pg*hr/mL to about 271.3 pg*hr/mL (e.g., from 173.60 pg*hr/mLto about 271.25 pg*hr/mL; or about 217 pg*hr/mL), as assessed at day 1;2) a corrected arithmetic mean peak plasma concentration (C_(avg[0-24]))of estradiol ranging from about 7.2 pg/mL to about 11.4 pg/mL (e.g.,from 7.25 pg/mL to about 11.33 pg/mL; or about 9.06 pg/mL), as assessedat day 1; 3) an unadjusted arithmetic mean area under the curve(AUC)₀₋₂₄ of estradiol ranging from about 137.5 pg*hr/mL to about 215.1pg*hr/mL (e.g., from 137.60 pg*hr/mL to about 215.00 pg*hr/mL; or about172 pg*hr/mL), as assessed at day 14; and 4) a corrected arithmetic meanpeak plasma concentration (C_(avg[0-24])) of estradiol ranging fromabout 5.7 pg/mL to about 9.0 pg/mL (e.g., from 5.72 pg/mL to about 8.94pg/mL; or about 7.15 pg/mL), as assessed at day 14.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estrone ranging fromabout 335.1 pg*hr/mL to about 523.8 pg*hr/mL (e.g., from 335.20 pg*hr/mLto about 523.75 pg*hr/mL; or about 419 pg*hr/mL), as assessed at day 1;2) a corrected arithmetic mean peak plasma concentration (C_(avg[0-24]))of estrone ranging from about 13.9 pg/mL to about 21.9 pg/mL (e.g., from14.00 pg/mL to about 21.88 pg/mL; or about 17.5 pg/mL), as assessed atday 1; 3) an unadjusted arithmetic mean area under the curve (AUC)₀₋₂₄of estrone ranging from about 343.1pg*hr/mL to about 536.2 pg*hr/mL(e.g., from 343.20 pg*hr/mL to about 536.25 pg*hr/mL; or about 429pg*hr/mL), as assessed at day 14; and 4) a corrected arithmetic meanpeak plasma concentration (C_(avg[0-24])) of estrone ranging from about14.3 pg/mL to about 22.4 pg/mL (e.g., from 14.32 pg/mL to about 22.38pg/mL; or about 17.9 pg/mL), as assessed at day 14.

In some embodiments, administration of a suppository comprising about 25μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estrone conjugatesranging from about 7,300.7 pg*hr/mL to about 11,407.6 pg*hr/mL (e.g.,from 7,300.80 pg*hr/mL to about 11,407.50 pg*hr/mL; or about 9,126pg*hr/mL), as assessed at day 1; 2) a corrected arithmetic mean peakplasma concentration (C_(avg[0-24])) of estrone conjugates ranging fromabout 303.9 pg/mL to about 475.1 pg/mL (e.g., from 304.00 pg/mL to about475.00 pg/mL; or about 380 pg/mL), as assessed at day 1; 3) anunadjusted arithmetic mean area under the curve (AUC)₀₋₂₄ of estroneconjugates ranging from about 7,943.9 pg*hr/mL to about 12,412.6pg*hr/mL (e.g., from 7,944.00 pg*hr/mL to about 12,412.50 pg*hr/mL; orabout 9,930 pg*hr/mL), as assessed at day 14; and 4) a correctedarithmetic mean peak plasma concentration (C_(avg[0-24])) of estroneconjugates ranging from about 331.1 pg/mL to about 517.4 pg/mL (e.g.,from 331.20 pg/mL to about 517.50 pg/mL; or about 414 pg/mL), asassessed at day 14.

In some embodiments, a suppository can comprise about 10 μg ofestradiol. In such cases, administration of the suppository to a patientcan provide, in a plasma sample from the patient, one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estradiol of about 12 pg*hr/mL to about 18 pg*hr/mL (e.g.,12.22 pg*hr/mL to about 17.98 pg*hr/mL); 2) a corrected geometric meanarea under the curve (AUC)₀₋₂₄ of estradiol of about 42 pg*hr/mL toabout 63 pg*hr/mL (e.g., 42.18 pg*hr/mL to about 62.02 pg*hr/mL); and 3)a corrected geometric mean time to peak plasma concentration (T_(max))of estradiol of about 1 hrs to about 3 hrs (e.g., 1.49 hrs to about 2.19hrs). In some embodiments, administration of the suppository to apatient provides, in a plasma sample from the patient, one or moreparameters selected from: 1) a corrected geometric mean peak plasmaconcentration (C_(max)) of estrone of about 4 pg*hr/mL to about 7pg*hr/mL (e.g., 4.38 pg*hr/mL to about 6.44 pg*hr/mL); 2) a correctedgeometric mean area under the curve (AUC)₀₋₂₄ of estrone of about 20pg*hr/mL to about 31 pg*hr/mL (e.g., 20.60 pg*hr/mL to about 30.30pg*hr/mL); and 3) a corrected geometric mean time to peak plasmaconcentration (T_(max)) of estrone of about 4 hrs to about 8 hrs (e.g.,4.99 hrs to about 7.34 hrs). In some embodiments, administration of thesuppository to a patient provides, in a plasma sample from the patient,one or more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 10 pg*hr/mLto about 16 pg*hr/mL (e.g., 10.34 pg*hr/mL to about 15.20 pg*hr/mL); 2)a corrected geometric mean area under the curve (AUC)₀₋₂₄ of estronesulfate of about 56 pg*hr/mL to about 84 pg*hr/mL (e.g., 56.61 pg*hr/mLto about 83.25 pg*hr/mL); and 3) a corrected geometric mean time to peakplasma concentration (T_(max)) of estrone sulfate of about 4 hrs toabout 7 hrs (e.g., 4.67 hrs to about 6.86 hrs).

In some embodiments, a suppository includes about 10 μg of estradiol. Insome such embodiments, administration of the suppository to a patientcan provide, in a plasma sample from the patient, a corrected geometricmean peak plasma concentration (C_(max)) of estradiol ranging from about4.7 pg/mL to about 7.6 pg/mL (e.g., 4.80 pg*hr/mL to about 7.50pg*hr/mL), as assessed at day 1. In some embodiments, administration ofa suppository comprising about 10 μg of estradiol to a patient canprovide, in a plasma sample from the patient, a corrected geometric meanpeak plasma concentration (C_(max)) of estradiol ranging from about 2.3pg*hr/mL to about 3.8 pg*hr/mL (e.g., 2.40 pg*hr/mL to about 3.75pg*hr/mL) of estradiol as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, a corrected geometric mean peak plasma concentration (C_(max))of estradiol of about 6.0 pg/mL, as assessed at day 1. In someembodiments, administration of a suppository comprising about 10 μg ofestradiol to a patient can provide, in a plasma sample from the patient,a corrected geometric mean peak plasma concentration (C_(max)) ofestradiol of about 3.0 pg/mL, as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, a corrected geometric mean area under the curve (AUC)₀₋₂₄ ofestradiol ranging from about 17.5 pg/mL to about 27.4 pg/mL (e.g., 17.52pg*hr/mL to about 27.37 pg*hr/mL), as assessed at day 1. In someembodiments, administration of a suppository comprising about 10 μg ofestradiol to a patient can provide, in a plasma sample from the patient,a corrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiolranging from about 10.9 pg*hr/mL to about 17.2 pg*hr/mL (e.g., 10.96pg*hr/mL to about 17.13 pg*hr/mL) of estradiol as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, a corrected geometric mean area under the curve (AUC)₀₋₂₄ ofestradiol of about 21.9 pg*hr/mL, as assessed at day 1. In someembodiments, administration of a suppository comprising about 10 μg ofestradiol to a patient can provide, in a plasma sample from the patient,a corrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiolof about 13.7 pg*hr/mL, as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, an average concentration (C_(avg)) of estradiol ranging fromabout 0.6 pg/mL to about 1.1 pg/mL (e.g., 0.64 pg/mL to about 1.0pg/mL), as assessed at day 1. In some embodiments, administration of asuppository comprising about 10 μg of estradiol to a patient canprovide, in a plasma sample from the patient, an average concentration(C_(avg)) of estradiol ranging from about 0.1 pg/mL to about 0.3 pg/mL(e.g., 0.16 pg/mL to about 0.25 pg/mL) of estradiol as assessed at day14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, an average concentration (C_(avg)) of estradiol of about 0.8pg/mL, as assessed at day 1. In some embodiments, administration of asuppository comprising about 10 μg of estradiol to a patient canprovide, in a plasma sample from the patient, an average concentration(C_(avg)) of estradiol of about 0.2 pg/mL, as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone conjugates rangingfrom about 72.1 pg/mL to about 112.8 pg/mL (e.g., 72.16 pg/mL to about112.75 pg/mL); and 2) an average concentration (C_(avg)) of estroneconjugates ranging from about 6.3 pg/mL to about 10.1 pg/mL (e.g., 6.40pg/mL to about 10.00 pg/mL) as assessed at day 1.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estrone conjugates of about90.2 pg/mL; and 2) an average concentration (C_(avg)) of estroneconjugates of about 8.0 pg/mL, as assessed at day 1.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estradiol ranging fromabout 110.3 pg*hr/mL to about 172.6 pg*hr/mL (e.g., from 110.40 pg*hr/mLto about 172.50 pg*hr/mL; or about 138 pg*hr/mL), as assessed at day 1;2) a corrected arithmetic mean peak plasma concentration (C_(avg[0-24]))of estradiol ranging from about 4.6 pg/mL to about 7.8 pg/mL (e.g., from4.61 pg/mL to about 7.20 pg/mL; or about 5.76 pg/mL), as assessed at day1; 3) an unadjusted arithmetic mean area under the curve (AUC)₀₋₂₄ ofestradiol ranging from about 87.9 pg*hr/mL to about 137.4 pg*hr/mL(e.g., from 88.00 pg*hr/mL to about 137.50 pg*hr/mL; or about 110pg*hr/mL), as assessed at day 14; and 4) a corrected arithmetic meanpeak plasma concentration (C_(avg[0-24])) of estradiol ranging fromabout 3.6 pg/mL to about 5.8 pg/mL (e.g., from 3.67 pg/mL to about 5.74pg/mL; or about 4.59 pg/mL), as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estrone ranging fromabout 370.3 pg*hr/mL to about 578.8 pg*hr/mL (e.g., from 370.40 pg*hr/mLto about 578.75 pg*hr/mL; or about 463 pg*hr/mL), as assessed at day 1;2) a corrected arithmetic mean peak plasma concentration (C_(avg[0-24]))of estrone ranging from about 15.4 pg/mL to about 24.2 pg/mL (e.g., from15.44 pg/mL to about 24.13 pg/mL; or about 19.3 pg/mL), as assessed atday 1; 3) an unadjusted arithmetic mean area under the curve (AUC)₀₋₂₄of estrone ranging from about 371.1 pg*hr/mL to about 580.1 pg*hr/mL(e.g., from 371.20 pg*hr/mL to about 580.00 pg*hr/mL; or about 464pg*hr/mL), as assessed at day 14; and 4) a corrected arithmetic meanpeak plasma concentration (C_(avg[0-24])) of estrone ranging from about15.4 pg/mL to about 24.2 pg/mL (e.g., from 15.44 pg/mL to about 24.13pg/mL; or about 19.3 pg/mL), as assessed at day 14.

In some embodiments, administration of a suppository comprising about 10μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estrone conjugatesranging from about 4,745.5 pg*hr/mL to about 7,414.9 pg*hr/mL (e.g.,from 4,745.60 pg*hr/mL to about 7,415.00 pg*hr/mL; or about 5,932pg*hr/mL), as assessed at day 1; 2) a corrected arithmetic mean peakplasma concentration (C_(avg[0-24])) of estrone conjugates ranging fromabout 197.5 pg/mL to about 308.8 pg/mL (e.g., from 197.60 pg/mL to about308.75 pg/mL; or about 247 pg/mL), as assessed at day 1; 3) anunadjusted arithmetic mean area under the curve (AUC)₀₋₂₄ of estroneconjugates ranging from about 7,182.3 pg*hr/mL to about 11,222.6pg*hr/mL (e.g., from 7,182.40 pg*hr/mL to about 11,222.50 pg*hr/mL; orabout 8,978 pg*hr/mL), as assessed at day 14; and 4) a correctedarithmetic mean peak plasma concentration (C_(avg[0-24])) of estroneconjugates ranging from about 299.1 pg/mL to about 467.6 pg/mL (e.g.,from 299.20 pg/mL to about 467.50 pg/mL; or about 374 pg/mL), asassessed at day 14.

In some embodiments, a suppository can comprise about 4 μg of estradiol.In such cases, administration of the suppository to a patient canprovide, in a plasma sample from the patient, one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estradiol of about 4 pg*hr/mL to about 8 pg*hr/mL; 2) acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiol ofabout 16 pg*hr/mL to about 26 pg*hr/mL; and 3) a corrected geometricmean time to peak plasma concentration (T_(max)) of estradiol of about0.25 hrs to about 2 hrs. In some embodiments, administration of thesuppository to a patient provides, in a plasma sample from the patient,one or more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone of about 1 pg*hr/mL to about 3pg*hr/mL; 2) a corrected geometric mean area under the curve (AUC)₀₋₂₄of estrone of about 8 pg*hr/mL to about 13 pg*hr/mL; and 3) a correctedgeometric mean time to peak plasma concentration (T_(max)) of estrone ofabout 1 hrs to about 4 hrs. In some embodiments, administration of thesuppository to a patient provides, in a plasma sample from the patient,one or more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 4 pg*hr/mL toabout 7 pg*hr/mL; 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone sulfate of about 22 pg*hr/mL to about 34 pg*hr/mL;and 3) a corrected geometric mean time to peak plasma concentration(T_(max)) of estrone sulfate of about 1 hrs to about 3 hrs.

In some embodiments, a suppository includes about 4 μg of estradiol. Insome such embodiments, administration of the suppository to a patientcan provide, in a plasma sample from the patient, one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estradiol ranging from about 2.0 pg/mL to about 3.3 pg/mL(e.g., 2.08 pg*hr/mL to about 3.25 pg*hr/mL); and 2) a correctedgeometric mean area under the curve (AUC)₀₋₂₄ of estradiol ranging fromabout 9.5 pg*hr/mL to about 15.1 pg*hr/mL (e.g., 9.60 pg*hr/mL to about15.0 pg*hr/mL), as assessed at day 1. In some embodiments,administration of a suppository comprising about 4 μg of estradiol to apatient can provide, in a plasma sample from the patient, one or moreparameters selected from: 1) a corrected geometric mean peak plasmaconcentration (C_(max)) of estradiol ranging from about 1.0 pg*hr/mL toabout 1.7 pg*hr/mL (e.g., 1.04 pg*hr/mL to about 1.63 pg*hr/mL) ofestradiol, and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estradiol ranging from about 5.7 pg*hr/mL to about 9.1pg*hr/mL (e.g., 5.76 pg*hr/mL to about 9.0 pg*hr/mL).

In some embodiments, administration of a suppository comprising about 4μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) a corrected geometricmean peak plasma concentration (C_(max)) of estradiol of about 2.6pg/mL; and 2) a corrected geometric mean area under the curve (AUC)₀₋₂₄of estradiol of about 12 pg*hr/mL, as assessed at day 1. In someembodiments, administration of a suppository comprising about 10 μg ofestradiol to a patient can provide, in a plasma sample from the patient,one or more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estradiol of about 1.3 pg/mL; 2) acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiol ofabout 7.2 pg*hr/mL, as assessed at day 14.

In some embodiments, administration of a suppository comprising about 4μg of estradiol to a patient provides, in a plasma sample from thepatient, a corrected geometric mean peak plasma concentration (C_(max))of estrone conjugates ranging from about 0.3 pg/mL to about 0.5 pg/mL(e.g., 0.32 pg/mL to about 0.5 pg/mL) as assessed at day 1.

In some embodiments, administration of a suppository comprising about 4μg of estradiol to a patient provides, in a plasma sample from thepatient, a corrected geometric mean peak plasma concentration (C_(max))of estrone conjugates of about 0.4 pg/mL as assessed at day 1.

In some embodiments, administration of a suppository comprising about 4μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estradiol ranging fromabout 73.3 pg*hr/mL to about 114.7 pg*hr/mL (e.g., from 73.36 pg*hr/mLto about 114.63 pg*hr/mL; or about 91.7 pg*hr/mL), as assessed at day 1;2) a corrected arithmetic mean peak plasma concentration (C_(avg[0-24]))of estradiol ranging from about 3.1 pg/mL to about 4.8 pg/mL (e.g., from3.14 pg/mL to about 4.90 pg/mL; or about 3.92 pg/mL), as assessed at day1; 3) an unadjusted arithmetic mean area under the curve (AUC)₀₋₂₄ ofestradiol ranging from about 69.7 pg*hr/mL to about 108.9 pg*hr/mL(e.g., from 69.76 pg*hr/mL to about 109.00 pg*hr/mL; or about 87.2pg*hr/mL), as assessed at day 14; and 4) a corrected arithmetic meanpeak plasma concentration (C_(avg[)o-24_(])) of estradiol ranging fromabout 2.8 pg/mL to about 4.6 pg/mL (e.g., from 2.90 pg/mL to about 4.54pg/mL; or about 3.63 pg/mL), as assessed at day 14.

In some embodiments, administration of a suppository comprising about 4μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estrone ranging fromabout 231.9 pg*hr/mL to about 362.4 pg*hr/mL (e.g., from 232.00 pg*hr/mLto about 362.50 pg*hr/mL; or about 290 pg*hr/mL), as assessed at day 1;2) a corrected arithmetic mean peak plasma concentration (C_(avg[0-24]))of estrone ranging from about 10.3 pg/mL to about 16.3 pg/mL (e.g., from10.40 pg/mL to about 16.25 pg/mL; or about 13 pg/mL), as assessed at day1; 3) an unadjusted arithmetic mean area under the curve (AUC)₀₋₂₄ ofestrone ranging from about 261.5 pg*hr/mL to about 408.8 pg*hr/mL (e.g.,from 261.60 pg*hr/mL to about 408.75 pg*hr/mL; or about 327 pg*hr/mL),as assessed at day 14; and 4) a corrected arithmetic mean peak plasmaconcentration (C_(avg[0-24])) of estrone ranging from about 10.8 pg/mLto about 17.1 pg/mL (e.g., from 10.88 pg/mL to about 17.00 pg/mL; orabout 13.6 pg/mL), as assessed at day 14.

In some embodiments, administration of a suppository comprising about 4μg of estradiol to a patient provides, in a plasma sample from thepatient, one or more parameters selected from: 1) an unadjustedarithmetic mean area under the curve (AUC)₀₋₂₄ of estrone conjugatesranging from about 4,062.3 pg*hr/mL to about 6,347.6 pg*hr/mL (e.g.,from 4,062.40 pg*hr/mL to about 6,347.50 pg*hr/mL; or about 5,078pg*hr/mL), as assessed at day 1; 2) a corrected arithmetic mean peakplasma concentration (C_(avg[0-24])) of estrone conjugates ranging fromabout 172.7 pg/mL to about 270.1 pg/mL (e.g., from 172.80 pg/mL to about270.00 pg/mL; or about 216 pg/mL), as assessed at day 1; 3) anunadjusted arithmetic mean area under the curve (AUC)₀₋₂₄ of estroneconjugates ranging from about 4,138.3 pg*hr/mL to about 6,466.3 pg*hr/mL(e.g., from 4,138.40 pg*hr/mL to about 6,466.25 pg*hr/mL; or about 5173pg*hr/mL), as assessed at day 14; and 4) a corrected arithmetic meanpeak plasma concentration (C_(avg[0-24])) of estrone conjugates rangingfrom about 172.7 pg/mL to about 270.1 pg/mL (e.g., from 172.80 pg/mL toabout 270.00 pg/mL; or about 216 pg/mL), as assessed at day 14.

A pharmaceutical composition provided herein can result in substantiallylocal delivery of estradiol. For example, plasma concentrations ofestradiol, estrone, and estrone sulfate measured in the plasma of apatient following administration of a pharmaceutical composition asprovided herein be statistically similar to those measured followingadministration of a placebo formulation (i.e., a similar formulationlacking the estradiol). Accordingly, in some embodiments, the plasmaconcentrations of estradiol, estrone, or estrone sulfate measuredfollowing administration of a pharmaceutical composition provided hereinmay be low compared to RLD formulations.

In some embodiments, a suppository can include about 1 μg to about 25 μgof estradiol. Upon administration the suppository to a patient, a plasmasample from the patient can provide a corrected geometric mean peakplasma concentration (C_(max)) of estradiol that is less than about 30pg*hr/mL. For example, administration of the suppository to a patientprovides a corrected geometric mean peak plasma concentration (C_(max))of estradiol that is less than about 18 pg*hr/mL. In some embodiments,administration of the suppository to a patient provides a correctedgeometric mean area under the curve (AUC)0-24 of estradiol that is lessthan about 112 pg*hr/mL. For example, administration of the suppositoryto a patient provides a corrected geometric mean area under the curve(AUC)0-24 of estradiol that is less than about 63 pg*hr/mL.

In some embodiments, administration of the suppository to a patientprovides a corrected geometric mean peak plasma concentration (C_(max))of estrone that is less than about 14 pg*hr/mL. For example,administration of the suppository to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estrone that isless than about 7 pg*hr/mL. In some embodiments, administration of thesuppository to a patient provides a corrected geometric mean area underthe curve (AUC)₀₋₂₄ of estrone that is less than about 65 pg*hr/mL. Forexample, administration of the suppository to a patient provides acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estrone thatis less than about 31 pg*hr/mL.

In some embodiments, administration of the suppository to a patientprovides a corrected geometric mean peak plasma concentration (C_(max))of estrone sulfate that is less than about 613 pg*hr/mL. For example,administration of the suppository to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estrone sulfatethat is less than about 16 pg*hr/mL. In some embodiments, administrationof the suppository to a patient provides a corrected geometric mean areaunder the curve (AUC)0-24 of estrone sulfate that is less than about5291 pg*hr/mL. For example, administration of the suppository to apatient provides a corrected geometric mean area under the curve(AUC)₀-24 of estrone sulfate that is less than about 84 pg*hr/mL.

In further aspects of this disclosure, capsule disintegration may bedetermined. In some embodiments, delivery vehicle disintegration orabsorption (presence or absence of the delivery vehicle afteradministration) at day 1 of treatment (for example, at 6 hours postfirst treatment) and at day 15 (for example, one day post the lasttreatment on day 14).

The pharmaceutical compositions can be formulated as described herein toprovide desirable pharmacokinetic parameters in a subject (e.g., afemale subject) to whom the composition is administered. In someembodiments, a pharmaceutical composition as described herein producesdesirable pharmacokinetic parameters for estradiol in the subject. Insome embodiments, a pharmaceutical composition as described hereinproduces desirable pharmacokinetic parameters for one or moremetabolites of estradiol in the subject, for example, estrone or totalestrone.

Following the administration of a composition comprising estradiol to asubject, the concentration and metabolism of estradiol can be measuredin a sample (e.g., a blood, serum, or plasma sample) from the subject.Estradiol is typically converted reversibly to estrone, and bothestradiol and estrone can be converted to the metabolite estriol. Inpostmenopausal women, a significant proportion of circulating estrogensexist as sulfate conjugates, especially estrone sulfate. Thus, estronecan be measured with respect to “estrone” amounts (excluding conjugatessuch as estrone sulfate) and “total estrone” amounts (including bothfree, or unconjugated, estrone and conjugated estrone such as estronesulfate).

The pharmaceutical compositions of this disclosure can be characterizedfor one or more pharmacokinetic parameters of estradiol or a metabolitethereof following administration of the composition to a subject or to apopulation of subjects. These pharmacokinetic parameters include AUC,C_(max), C_(avg), and T_(max). AUC is a determination of the area underthe curve (AUC) plotting the blood, serum, or plasma concentration ofdrug along the ordinate (Y-axis) against time along the abscissa(X-axis). AUCs are well understood, frequently used tools in thepharmaceutical arts and have been extensively described. C_(max) is wellunderstood in the art as an abbreviation for the maximum drugconcentration in blood, serum, or plasma of a subject. T_(max) is wellunderstood in the art as an abbreviation for the time to maximum drugconcentration in blood, serum, or plasma of a subject.

In some embodiments, one or more pharmacokinetic parameters, e.g., AUC,C_(max), C_(avg), or T_(max), is measured for estradiol. In someembodiments, one or more pharmacokinetic parameters, e.g., AUC, C_(max),C_(avg), or T_(max), is measured for estrone. In some embodiments, oneor more pharmacokinetic parameters, e.g., AUC, C_(max), C_(avg), orT_(max), is measured for total estrone. Any pharmacokinetic parametercan be a “corrected” parameter, wherein the parameter is determined as achange over a baseline level.

Any of a variety of methods can be used for measuring the levels ofestradiol, estrone, or total estrone in a sample, includingimmunoassays, mass spectrometry (MS), high performance liquidchromatography (HPLC) with ultraviolet fluorescent detection, liquidchromatography in conjunction with mass spectrometry (LC-MS), tandemmass spectrometry (MS/MS), and liquid chromatography-tandem massspectrometry (LC-MS/MS). In some embodiments, the levels of estradiol,estrone, or total estrone are measured using a validated LC-MS/MSmethod. Methods of measuring hormone levels are well described in theliterature.

Statistical Measurements

According to embodiments, pharmacokinetics of the pharmaceuticalcomposition disclosed herein are measured using statistical analysis.According to embodiments, Analysis of Variance (“ANOVA”) or Analysis ofCoVariance (“ANCOVA”) are used to evaluate differences between a patientreceiving treatment with a pharmaceutical composition comprising anactive pharmaceutical composition (for example, a pharmaceuticalcomposition comprising estradiol) and a patient receiving treatment witha placebo (for example, the same pharmaceutical composition but withoutestradiol) or a reference drug. A person of ordinary skill in the artwill understand how to perform statistical analysis of the datacollected.

VIII. EXAMPLES

The following examples are of pharmaceutical compositions, deliveryvehicles, and combinations thereof. Methods of making are alsodisclosed. Data generated using the pharmaceutical compositionsdisclosed herein are also disclosed.

Example 1 Pharmaceutical Composition

In embodiments, estradiol is procured and combined with one or morepharmaceutically acceptable solubilizing agents. The estradiol ispurchased as a pharmaceutical grade ingredient, often as micronizedestradiol, although other forms can also be used. In embodiments, thepharmaceutical composition includes estradiol in a dosage strength offrom about 1 μg to about 50 μg. In embodiments, the pharmaceuticalcomposition includes 10 μg of estradiol. In embodiments, thepharmaceutical composition includes 25 μg of estradiol.

In embodiments, the estradiol is combined with pharmaceuticallyacceptable solubilizing agents, and, optionally, other excipients, toform a pharmaceutical composition. In embodiments, the solubilizingagent is one or more of CAPMUL MCM, MIGLYOL 812, GELUCIRE 39/01,GELUCIRE 43/01, GELUCIRE 50/13, and TEFOSE 63.

GELUCIRE 39/01 and GELUCIRE 43/01 each have an HLB value of 1. GELUCIRE50/13 has an HLB value of 13. TEFOSE 63 has an HLB value of between 9and 10.

Various combinations of pharmaceutically acceptable solubilizing agentswere combined with estradiol and examined as shown in Table 1.

TABLE 1 Capmul MCM (“MCM”), Gelucire 39/01 (“39/01”), Gelucire 43/01(“43/01”), Gelucire 50/13(“50/13”), and Tefose (“Tefose 63”) PhysicalPhysical state state @ @ 37° C. Melting Dispersion Vehicle Room after~30 Viscosity Time @ in water # system Ratio Temp. minutes (cps) 37° C.37° C. 1 MCM:39/01 8:2 Solid Clear liquid  50 @ 37° C. Start: 6 minSmall oil Finish: 12 min drops on top 2 MCM:39/01 7:3 Solid Clear liquidStart: 9 min Finish: 19 min 3 MCM:39/01 6:4 Solid Clear liquid Start: 20min Finish: 32 min 4 MCM:43/01 8:2 Solid Liquid with solid particles 5MCM:43/01 7:3 Solid Liquid with solid particles 6 MCM:50/13 9:1 Liquid/Liquid/ 140@ 25° C. Clear after Uniformly cloudy cloudy 20 min cloudydispersion 7 MCM:50/13 8:2 Liquid/ Liquid/ 190@ 25° C. Uniformly cloudycloudy cloudy dispersion 8 MCM:50/13 7:3 Semisolid Semisolid 9MCM:TEFOSE 9:1 Semisolid Liquid/ 150@ 25° C. Start: 1 min Uniformly 63cloudy Finish: 5 min cloudy dispersion 10 MCM:TEFOSE 8:2 SemisolidSemisolid 240@ 25° C. Uniformly 63 cloudy dispersion 11 MCM:TEFOSE 7:3Semisolid Semisolid 380@ 25° C. Semisolid Uniformly 63 after 30 mincloudy at 37° C., dispersion doesn't melt at 41° C. 12 MIGLYOL 9:1Semisolid Semisolid 140@ 25° C. 2 phases, oil 812:50/13 on top 13MIGLYOL 9:1 Liquid/ Liquid/  90@ 25° C. Start: 1 min 2 phases, oil812:TEFOSE cloudy cloudy Finish: 5 min on top 63

Pharmaceutical compositions in Table 1 that were liquid or semisolid atroom temperature were tested using a Brookfield viscometer (BrookfieldEngineering Laboratories, Middleboro, Mass.) at room temperature.Pharmaceutical compositions appearing in Table 1 that were solid atambient temperature were tested using a Brookfield viscometer at 37° C.

Pharmaceutical compositions appearing in Table 1 that were solid at roomtemperature were assessed at 37° C. to determine their meltingcharacteristics. The viscosity of the gels can be important duringencapsulation of the formulation. For example, in some cases, it isnecessary to warm the formulation prior to filing of the gelatincapsules. In addition, the melting characteristics of the compositioncan have important implications following administration of theformulation into the body. For example, in some embodiments, theformulation will melt at temperatures below about 37° C. PharmaceuticalComposition 11 (Capmul MCM/Tefose 63), for example, did not melt at 37°C. or 41° C.

A dispersion assessment of the pharmaceutical compositions appearing inTable 1 was performed. The dispersion assessment was performed bytransferring 300 mg of each vehicle system in 100 mL of 37° C. water,without agitation, and observing for mixing characteristics. Resultsvaried from formation of oil drops on the top to separation of phases touniform, but cloudy dispersions. Generally speaking, it is believed thatformulations able to readily disperse in aqueous solution will havebetter dispersion characteristics upon administration. It wassurprisingly found, however, as shown below in Examples 7-9, thatformulations that did not readily disperse in aqueous solution (e.g.,Formulation 13) and instead formed two phases upon introduction to theaqueous solution were found to be the most effective when administeredto the human body.

Example 2 Delivery Vehicle

In embodiments, the pharmaceutical composition is delivered in a gelatincapsule delivery vehicle. The gelatin capsule delivery vehicle includes,for example, gelatin (e.g., Gelatin, NF (150 Bloom, Type B)), hydrolyzedcollagen (e.g., GELITA®, GELITA AG, Eberbach, Germany), glycerin,sorbitol special, or other excipients in proportions that are well knownand understood by persons of ordinary skill in the art. Sorbitol specialmay be obtained commercially and may tend to act as a plasticizer andhumectant.

A variety of delivery vehicles were developed, as show in Table 2, GelsA through F. In Table 2, each delivery vehicle A through F differs inthe proportion of one or more components.

TABLE 2 Gelatin Capsule Delivery Vehicles A B C D E F Ingredient % w/w %w/w % w/w % w/w % w/w % w/w Gelatin, NF (150 Bloom, Type B) 41.0 41.041.0 41.0 43.0 43.0 Glycerin 99.7%, USP 6.0 6.0 6.0 6.0 18.0 18.0Sorbitol Special, USP 15.0 15.0 15.0 15.0 GELITA ® (hydrolyzed collagen)3 3.0 Citric acid 0.1 0.5 1 0.1 Purified Water 35.0 37.9 37.5 37.0 36.038.9 Total 100.0 100.0 100.0 100.0 100.0 100.0 Dissolution gel strips,Avg of 3 48 min 50 min 75 min 70 min (500 mL DH2O, 50 rpm @ 37° C.) (42,45, 58) (50, 51, 50) (76, 75, 74) (70, 71, 70) Dissolution gel strips,Avg of 3 70 min 78 min 82 min (500 mL pH 4 buffer, 50 rpm @ 37° C.)

Each delivery vehicle A through F was prepared at a temperature rangefrom about 45° C. to about 85° C. Each molten delivery vehicle A throughF was cast into a film, dried, and cut into strips. The strips were cutinto uniform pieces weighing about 0.5 g, with about 0.5 mm thickness.Strips were placed into a USP Type 2 dissolution vessel in either wateror pH 4 buffer solution and the time for them to completely dissolve wasrecorded (see Table 2). Delivery vehicle A had the fastest dissolutionin both water and pH 4 buffer solution.

Example 3 Pharmaceutical Compositions and Delivery Vehicle

Various combinations of the pharmaceutical compositions from Table 1 andfrom Table 2 were prepared. The combinations are shown in Table 3.

TABLE 3 Delivery Trial Pharmaceutical Composition Ratio Batch Size gVehicle 1 MCM:39/01 8:2 750 A 2 MCM:50/13 8:2 750 A 3 MCM:TEFOSE 63 8:2750 A 4 MCM:TEFOSE 63 8:2 750 B 5 MIGLYOL 812:TEFOSE 63 9:1 750 A

Each aliquot of the pharmaceutical compositions of Table 3 about 300 mgto about 310 mg. Batch size was as listed in Table 3. To encapsulate thevehicle system, each 300 mg to about 310 mg pharmaceutical compositionaliquot was encapsulated in about 200 mg of the gelatin capsule deliveryvehicle. Thus, for example, in Trial 1, the pharmaceutical compositiondenoted by MCM:39/01 was encapsulated in gelatin capsule deliveryvehicle A for a total encapsulated weight of about 500 mg to about 510mg. The aliquot size is arbitrary depending on the concentration of theestradiol and the desired gelatin capsule delivery vehicle size.Artisans will readily understand how to adjust the amount of estradiolin the pharmaceutical composition to accommodate a given size ofdelivery vehicle, when the delivery vehicle encapsulates thepharmaceutical composition.

Example 4 Estradiol Solubility

In various experiments, solubilizing agents were tested to determinewhether they were able to solubilize 2 mg of estradiol for a totalpharmaceutical composition weight of 100 mg. The solubilizing agentswere considered suitable if estradiol solubility in the solubilizingagent was greater than or equal to about 20 mg/g. Initial solubility wasmeasured by dissolving micronized estradiol into various solubilizingagents until the estradiol was saturated (the estradiol/solubilizingagent equilibrated for three days), filtering the undissolved estradiol,and analyzing the resulting pharmaceutical composition for estradiolconcentration by HPLC.

TABLE 4 Solubility of Solubilizing Agents (*denotes literaturereference) Ingredient Solubility (mg/g) PEG 400 105*  Propylene Glycol75* Polysorbate 80 36* TRANSCUTOL HP 141  CAPMUL PG8  31.2

Example 5 Pharmaceutical Compositions

The following pharmaceutical compositions are contemplated.

Gel Mass

Ingredient % w/w Qty/Batch (kg) Gelatin 150 Bloom Limed Bone, NF 41.0082.00 Hydrolyzed Gelatin 3.00 6.00 Glycerin 99.7% 6.00 12.00 SorbitolSpecial, NF 15.00 30.00 Opatint White G-18006 1.20 2.40 Opatine RedDG-15001 0.06 0.12 Purified Water, USP 33.74 67.48 Total 100.00 200.00Kg

Pharmaceutical Composition 1: 10 μg Estradiol

Qty/ Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydratemicronized, USP 0.010 0.003 0.10 g CAPMUL ® MCM, NF (Glyceryl 240.079.997 2.40 kg Caprylate/Caprate or Medium Chain Mono- and Diglycerides)GELUCIRE ® 50/13 (stearoyl polyoxyl- 60.0 20.0 600.0 g 32 glycerides NF)Total 300.0 100.0 3.0 kg

Pharmaceutical Composition 2: 10 μg Estradiol

Qty/ Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydratemicronized, USP 0.010 0.003 0.10 g MIGLOYL ® 812 (medium chain 270.089.997 2.70 kg triglyceride) TEFOSE ® 63 (mixture of PEG-6 30.0 10.0300.0 g stearate or ethylene glycol palmitostearate or PEG-32 stearate;polyoxyl 6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0100.0 3.00 kg

Pharmaceutical Composition 3: 25 μg estradiol

Qty/ Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydratemicronized, USP 0.026* 0.009 0.26 g MIGLOYL ® 812 (medium chain 270.089.991 2.70 kg triglyceride) TEFOSE ® 63 (mixture of PEG-6 30.02 10.0300.0 g stearate or ethylene glycol palmitostearate or PEG-32 stearate;polyoxyl 6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0100.0 3.00 kg *1.0 mg estradiol is equivalent to 1.03 mg estradiolhemihydrate

Pharmaceutical Composition 4: 4 μg Estradiol

Qty/ Qty/Batch Capsule (alternate Ingredients (mg) % w/w batch size)Estradiol hemihydrate micronized, 0.0041* 0.001 0.041 g USP (0.615 g)MIGLOYL ® 812 (medium chain 269.99 89.999 2700.0 g triglyceride) (40.50kg) TEFOSE ® 63 (mixture of PEG-6 30.0 10.0 300.0 g stearate or ethyleneglycol (4.50 kg) palmitostearate or PEG-32 stearate; polyoxyl 6 andpolyoxyl 32 palmitostearate/glycol stearate) Total 300.0 100.0 3000.0 g45.0 kg *1.0 mg estradiol is equivalent to 1.03 mg estradiol hemihydrate

Pharmaceutical Composition 5: 10 μg Estradiol

Qty/ Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydratemicronized, USP 0.0103* 0.003 1.545 g MIGLOYL ® 812 (medium chain 269.9989.997 40.5 kg triglyceride) TEFOSE ® 63 (mixture of PEG-6 30.0 10.04.50 kg stearate or ethylene glycol palmitostearate or PEG-32 stearate;polyoxyl 6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0100.0 45.00 kg *1.0 mg estradiol is equivalent to 1.03 mg estradiolhemihydrate

Pharmaceutical Composition 6: 25 μg Estradiol

Qty/ Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydratemicronized, USP 0.026* 0.009 3.90 g MIGLOYL ® 812 (medium chain 269.9789.991 40.50 kg triglyceride) TEFOSE ® 63 (mixture of PEG-6 30.0 10.04.50 kg stearate or ethylene glycol palmitostearate or PEG-32 stearate;polyoxyl 6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0100.0 45.00 kg *1.0 mg estradiol is equivalent to 1.03 mg estradiolhemihydrate

Pharmaceutical Composition 7: Placebo

Qty/ Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydratemicronized, USP 0.00 0.00 0.00 g MIGLOYL ® 812 (medium chain 270.0 90.040.5 kg triglyceride) TEFOSE ® 63 (mixture of PEG-6 30.0 10.0 4.5 kgstearate or ethylene glycol palmitostearate or PEG-32 stearate; polyoxyl6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0 100.03000.0 g

In the Examples below, TX-004HR is Pharmaceutical Compositions 4, 5, and6 (TX-004HR 4 μg, TX-004HR 10 μg, and TX-004HR 25 μg) compared toPharmaceutical Composition 7.

Example 6 Process

FIG. 1 illustrates an embodiment of a method making pharmaceuticalcomposition comprising estradiol solubilized in CapmulMCM/Geluciresolubilizing agent encapsulated in a soft gelatin delivery vehicle 100.In operation 102, the CapmulMCM is heated to 40° C.±5° C. Heating may beaccomplished through any suitable means. The heating may be performed inany suitable vessel, such as a stainless steel vessel. Otherpharmaceutical compositions can be made using the same general method bysubstituting various excipients, including the solubilizing agent.

In operation 104, GELUCIRE is mixed with the CapmulMCM to form thefinished solubilizing agent. As used herein, any form of GELUCIRE may beused in operation 104. For example, one or more of GELUCIRE 39/01,GELUCIRE 43/01, GELUCIRE 50/13 may be used in operation 104. Mixing isperformed as would be known to persons of ordinary skill in the art, forexample by impeller, agitator, stirrer, or other like devices used tomix pharmaceutical compositions. Operation 104 may be performed under aninert or relatively inert gas atmosphere, such as nitrogen gas. Mixingmay be performed in any vessels that are known to persons of ordinaryskill in the art, such as a stainless steel vessel or a steel tank.

In operation 106 estradiol is mixed into the solubilizing agent. Inembodiments, the estradiol in micronized when mixed into thesolubilizing agent. In other embodiments, the estradiol added is in anon-micronized form. Mixing may be facilitated by an impeller, agitator,stirrer, or other like devices used to mix pharmaceutical compositions.Operation 106 may be performed under an inert or relatively inert gasatmosphere, such as nitrogen gas.

In embodiments, however, the addition of estradiol may be performedprior to operation 104. In that regard, operations 104 and 106 areinterchangeable with respect to timing or can be performedcontemporaneously with each other.

In operation 110, the gelatin delivery vehicle is prepared. Any of thegelatin delivery vehicles described herein may be used in operation 110.In embodiments, gelatin, hydrolyzed collagen, glycerin, and otherexcipients are combined at a temperature range from about 45° C. toabout 85° C. and prepared as a film. Mixing may occur in a steel tank orother container used for preparing gelatin delivery vehicles. Mixing maybe facilitated by an impellor, agitator, stirrer, or other devices usedto combine the contents of gelatin delivery vehicles. Operation 110 maybe performed under an inert or relatively inert gas atmosphere, such asnitrogen gas. In embodiments, the gelatin delivery vehicle mixture isdegassed prior to being used to encapsulate the pharmaceuticalcomposition.

In operation 112, the gelatin delivery vehicle encapsulates thepharmaceutical composition, according to protocols well known to personsof ordinary skill in the art. In operation 112, a soft gelatin capsuledelivery vehicle is prepared by combining the pharmaceutical compositionmade in operation 106 with the gelatin delivery vehicle made inoperation 110. The gelatin may be wrapped around the material, partiallyor fully encapsulating it or the gelatin can also be injected orotherwise filled with the pharmaceutical composition made in operation106.

In embodiments, operation 112 is completed in a suitable die to providea desired shape. Vaginal soft gel capsules may be prepared in a varietyof geometries. For example, vaginal soft gel capsules may be shaped as atear drop, a cone with frustoconical end, a cylinder, a cylinder withlarger “cap” portion as illustrated in FIG. 2, or other shapes suitablefor insertion into the vagina. The resulting pharmaceutical compositionencapsulated in the soft gelatin delivery vehicle may be inserteddigitally or with an applicator.

Example 7 Study of Estradiol Pharmaceutical Composition on theImprovement of Vulvovaginal Atrophy (VVA)

The objective of this study was designed to evaluate the efficacy andsafety of a pharmaceutical composition comprising 10 μg estradiol (i.e.,Pharmaceutical Composition 2) in treating moderate to severe symptoms ofVVA associated with menopause after 14 days of treatment, and toestimate the effect size and variability of vulvovaginal atrophyendpoints. In addition, the systemic exposure to estradiol from singleand multiple doses of the pharmaceutical composition was investigated.

This study was a phase 1, randomized, double-blind, placebo-controlledtrial to evaluate safety and efficacy of the pharmaceutical compositionin reducing moderate to severe symptoms of vaginal atrophy associatedwith menopause and to investigate the systemic exposure to estradiolfollowing once daily intravaginal administrations of a pharmaceuticalcomposition for 14 days.

Postmenopausal subjects who met the study entry criteria were randomizedto one of two treatment groups (pharmaceutical composition or placebo).During the screening period subjects were asked to self-assess thesymptoms of VVA, including vaginal dryness, vaginal or vulvar irritationor itching, dysuria, vaginal pain associated with sexual activity, andvaginal bleeding associated with sexual activity. Subjects with at leastone self-assessed moderate to severe symptom of VVA identified by thesubject as being most bothersome to her were eligible to participate inthe study.

Clinical evaluations were performed at the following time points:

-   -   Screening Period (up to 28 days);    -   Visit 1—Randomization/Baseline (day 1);    -   Visit 2—Interim (day 8); and    -   Visit 3—End of the treatment (day 15).

Eligible subjects were randomized in a 1:1 ratio to receive eitherpharmaceutical composition comprising estradiol 10 μg or a matchingplacebo vaginal softgel capsule, and self-administered their first doseof study medication at the clinical facility under the supervision ofthe study personnel. Serial blood samples for monitoring of estradiollevel were collected at 0.0, 1.0, 3.0, and 6.0 hours relative to firstdose administration on day 1. Subjects remained at the clinical siteuntil completion of the 6-hour blood draw and returned to clinicalfacility for additional single blood draws for measurement of estradiolconcentration on day 8 (before the morning dose) and day 15. Subjectswere provided with enough study medication until the next scheduledvisit and were instructed to self-administer their assigned studytreatment once a day intravaginally at approximately the same time (±1hour) every morning. Each subject was provided with a diary in which shewas required to daily record investigational drug dosing dates andtimes. Subjects returned to clinical facility on day 8 for interim visitand on day 15 for end of treatment assessments and post studyexaminations. Capsule disintegration state was assessed by theinvestigator at day 1 (6 hours post-dose) and day 15.

The study involved a screening period of up to 28 days beforerandomization and treatment period of 14 days. Selection of dosagestrength (estradiol 10 μg) and treatment regimen (once daily for twoweeks) was based on the FDA findings on safety and efficacy of the RLD.

Number of Subjects (Planned and Analyzed)

Up to 50 (25 per treatment group) postmenopausal female subjects 40 to75 years old with symptoms of moderate to severe VVA were randomized. 50subjects were enrolled, 48 subjects completed the study, and 48 subjectswere analyzed.

Diagnosis and Main Criteria for Inclusion

Fifty female subjects were enrolled in the study. Post-menopausal femalesubjects 40 to 75 years of age, with a mean age was 62.3 years wereenrolled. Subjects' mean weight (kg) was 71.2 kg with a range of44.5-100 kg. Subjects' mean height (cm) was 162.6 cm with a range of149.9-175.2 cm, and the mean BMI (kg/m²) was 26.8 kg/m² with a range of19-33 kg/m². Criteria of inclusion in the study included:self-identification of at least one moderate to severe symptom of VVA,for example, vaginal dryness, dyspareunia, vaginal or vulvar irritation,burning, or itching, dysuria, vaginal bleeding associated with sexualactivity, that was identified by the subject as being most bothersome toher; ≤5% superficial cells on vaginal smear cytology; vaginal pH>5.0;and estradiol level ≤50 pg/mL. Subject who were judged as being inotherwise generally good health on the basis of a pre-study physicalexamination, clinical laboratory tests, pelvic examination, andmammography were enrolled.

Estradiol 10 μg or Placebo, Dose, and Mode of Administration

Subjects were randomly assigned (in 1:1 allocation) to self-administerone of the following treatments intravaginally once daily for 14 days:

-   -   Treatment A: The pharmaceutical composition of Example 5        (Pharmaceutical Composition 2: 10 μg estradiol); or    -   Treatment B: Placebo vaginal softgel capsule, containing the        same formulation as Treatment A, except for the 10 μg of        estradiol.

The estradiol formulation was a tear drop shaped light pink soft gelcapsule. Treatment B had the same composition, appearance, and route ofadministration as the Treatment A, but contained no estradiol.

Duration of Treatment

The study involved a screening period of up to 28 days beforerandomization and a treatment period of 14 days.

Criteria for Evaluation

Efficacy Endpoints:

-   -   Change from baseline (screening) to day 15 in the Maturation        Index (percent of parabasal vaginal cells, superficial vaginal        cells, and intermediate vaginal cells) of the vaginal smear.        Data for this endpoint are shown in Tables 6-8.    -   Change from baseline (screening) to day 15 in vaginal pH. Data        for this endpoint are shown in Table 9.    -   Change from baseline (randomization) to day 15 in severity of        the most bothersome symptoms: (1) vaginal dryness; (2) vaginal        or vulvar irritation, burning, or itching; (3) dysuria; (4)        dyspareunia; (5) vaginal bleeding associated with sexual        activity. Data for this endpoint are shown in Tables 13 and 15.    -   Change from baseline (randomization) to day 15 in investigator's        assessment of the vaginal mucosa. Data for this endpoint are        shown in Tables 18-21.

Unless otherwise noted, the efficacy endpoints were measured as achange-from Visit 1—Randomization/Baseline (day 1) to Visit 3—End of thetreatment (day 15), except for vaginal bleeding which was expressed aseither treatment success or failure.

Other endpoints include:

-   -   Vital signs, weight, changes in physical exam, pelvic and breast        exam, and adverse events were evaluated as part of the safety        endpoints.    -   Concentration of estradiol at each sampling time.    -   Peak concentration of estradiol on day 1 and sampling time at        which peak occurred.    -   Delivery vehicle disintegration to measure the amount of        residual delivery vehicle remains in the vagina post treatment.

Results from the assessment of plasma concentrations of estradiol arepresented in Table 5.

TABLE 5 Safety Results: The descriptive statistics for Day 1 plasmaestradiol C_(max) and T_(max) are provided below. Estradiol 10 μgPlacebo C_(max) T_(max) C_(max) T_(max) N 24 24 26 26 Mean ± SD 30.7 ±7.47 2.12 ± 1.73 27.5 ± 17.26 4.00 ± 2.68 Geometric 29.9 — 24.7 — MeanMedian 29.8 1.00 22.1 6.00 Min, Max 19.7, 52.3 1.00, 6.00 15.1, 90.00.00, 6.00 CV % 24.3% 81.3% 62.9% 67.1%

Maturation Index Results

Vaginal cytology data was collected as vaginal smears from the lateralvaginal walls according to standard procedures to evaluate vaginalcytology at screening and Visit 3—End of treatment (day 15). The changein the Maturation Index was assessed as a change in cell compositionmeasured at Visit 1—Baseline (day 1) compared to the cell compositionmeasured at Visit 3—End of treatment (day 15). The change in percentageof superficial, parabasal, and intermediate cells obtained from thevaginal mucosal epithelium from a vaginal smear was recorded. Resultsfrom these assessments are presented in Tables 6, 7, and 8.

TABLE 6 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in the Maturation Index of the Vaginal Smear(Percent Parabasal Cells) Difference Estradiol Between 10 μg vs.Estradiol Treatment 90% CI for Placebo Population Statistics 10 μgPlacebo Means Difference P-value Intent-to-Treat N 24  24 — — — Least-−54.4 −4.80 −49.6 (−60.4, −38.8) <0.0001 Squares Mean Mean ± SD −53.8 ±39.7 −5.4 ± 22.3 — — — Median −60.0 −5.0 — — — Min, Max −100.0, 0.0−60.0, 60.0 — — — ¹ Confidence interval for the estradiol 10 μg-Placebofrom ANCOVA with treatment as a fixed effect and baseline as acovariate. ²P-value for treatment comparison from ANCOVA with treatmentas a fixed effect and baseline as a covariate.

TABLE 7 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in the Maturation Index of the Vaginal Smear(Superficial Cells) Difference Estradiol 10 μg Between vs. EstradiolTreatment 90% CI for Placebo P- Population Statistics 10 μg PlaceboMeans Difference value Intent-to-Treat N 24 24 — — — Least- 35.2 8.7526.5 (15.4, 37.6) 0.0002 Squares Mean Mean ± SD 35.2 ± 26.4 8.8 ± 18.7 —— — Median 40.0 0.0 — — — Min, Max 0.0, 80.0 0.0, 90.0 — — — ¹Confidenceinterval for the estradiol 10 μg-Placebo from ANOVA with treatment as afixed effect. ²P-value for treatment comparison from ANOVA withtreatment as a fixed effect.

TABLE 8 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in the Maturation Index of the Vaginal Smear(Intermediate Cells) Difference Estradiol 10 μg Between vs. EstradiolTreatment 90% CI for Placebo P- Population Statistics 10 μg PlaceboMeans Difference value² Intent-to-Treat N 24 24 — — — Least- 18.7 −3.5422.3 (11.1, 33.5) 0.0017 Squares Mean Mean ± SD 18.5 ± 42.7 −3.3 ± 21.6— — — Median 22.5 −5.0 — — — Min, Max −60.0, 100.0 −60.0, 20.0 — — —¹Confidence interval for the estradiol 10 μg-Placebo from ANCOVA withtreatment as a fixed effect and baseline as a covariate. ²P-value fortreatment comparison from ANCOVA with treatment as a fixed effect andbaseline as a covariate.

Change in pH Results

Vaginal pH was measured at Screening and Visit 3—End of treatment (day15). The pH measurement was obtained by pressing a pH indicator stripagainst the vaginal wall. The subjects entering the study were requiredto have a vaginal pH value greater than 5.0 at screening. pH values wererecorded on the subject's case report form. The subjects were advisednot to have sexual activity and to refrain from using vaginal douchingwithin 24 hours prior to the measurement. Results from these assessmentsare presented in Table 9.

TABLE 9 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in Vaginal pH Estradiol 10 μg Difference vs.Estradiol Between 90% CI for Placebo P- Population Statistics 10 μgPlacebo Treatment Means Difference¹ value² Intent-to-Treat N 24 24 — — —Least- −0.974 −0.339 −0.635 (−0.900, −0.368) 0.0002 Squares Mean Mean ±SD −0.917 ± 0.686 −0.396 ± 0.659 — — — Median −1.00 −0.500 — — — Min,Max −2.00, 0.500 −1.50, 0.500 — — — ¹Confidence interval for theestradiol 10 μg-Placebo from ANCOVA with treatment as a fixed effect andbaseline as a covariate. ²P-value for treatment comparison from ANCOVAwith treatment as a fixed effect and baseline as a covariate.

Most Bothersome Symptoms Data

Subjects were asked to specify the symptom that she identified as the“most bothersome symptom.” During the screening period all of thesubjects were provided with a questionnaire to self-assess the symptomsof VVA: (1) vaginal dryness; (2) vaginal or vulvar irritation, burning,or itching; (3) dysuria; (4) dyspareunia; (5) vaginal bleedingassociated with sexual activity. Each symptom, with the exception ofvaginal bleeding associated with sexual activity, was measured on ascale of 0 to 3, where 0=none, 1=mild, 2=moderate, and 3=severe. Vaginalbleeding associated with sexual activity was measured in a binary scale:N=no bleeding; Y=bleeding. The subject's responses were recorded. Allrandomized subjects were also provided a questionnaire to self-assessthe symptoms of VVA at Visit 1—Randomization/Baseline (day 1) and atVisit 3—End of the treatment (day 15). Subjects recorded theirself-assessments daily in a diary and answers were collected on days 8and 15 (end of treatment). Pre-dose evaluation results obtained at Visit1 were considered as baseline data for the statistical analyses. Datafrom these assessments are presented in Tables 10 and 11.

TABLE 10 Baseline Characteristics for Vaginal Atrophy Symptoms (ITTPopulation) Estradiol 10 μg Estradiol vs. Placebo VVA Symptom Statistics10 μg Placebo P-value¹ Vaginal dryness N of 24 24 — Subjects Mean 2.2922.375 0.68231 Vaginal or vulvar ir- N of 24 24 —ritation/burning/itching Subjects Mean 0.875 1.333 0.08721 Pain, burningor N of 24 24 — stinging when Subjects urinating Mean 0.583 0.6250.87681 Vaginal pain asso- N of 12 12 — ciated with sexual Subjects²activity Mean 2.083 2.333 0.54281 Vaginal bleeding N of 12 12 associatedwith Subjects² sexual activity Percent³ 25.00 33.33 0.31463 ¹P-value tortreatment comparison from ANOVA/ANCOVA with treatment as a fixed effectand Baseline as a covariate when appropriate. ²N = number of subjectssexually active at baseline. ³Percent of subjects with bleeding,evaluated using Fisher's Exact Test.

TABLE 11 Additional Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Severity of Vaginal Atrophy SymptomsLeast-Squares Difference Mean Between Estradiol 10 μg StatisticalEstradiol Treatment 90% CI for vs. Placebo Symptom Method¹ 10 μg PlaceboMeans Difference² P-value Vaginal dryness ANCOVA 0.980 0.729 0.251−0.706, 0.204) 0.3597 Vaginal or vulvar ANCOVA 0.694 0.514 0.180 −0.549,0.189) 0.4159 Irritation/burning/ itching Pain/Burning/ ANCOVA 0.3910.359 0.032 −0.263, 0.200) 0.8185 Stinging (Urination) Vaginal painANOVA 0.800 0.500 0.300 −1.033, 0.433) 0.4872 associated with sexualactivity ¹ANOVA model contained a fixed effect for treatment. ANCOVAadded baseline as a covariate to the model. ²Confidence interval for thedifference between estradiol 10 μg and Placebo treatment least-squaresmeans.

Changes to the most bothersome symptom from the baseline was scoredaccording to the evaluation of VVA symptoms generally set forth above.Tables 13 and 14 show a comparison between the pharmaceuticalcomposition 1 and placebo generally for most bothersome symptom andvaginal atrophy symptom. It is noteworthy to point out that thesemeasurement demonstrated a trend of improvement, though notstatistically significant, at day 15.

TABLE 13 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Severity of the Most Bothersome VVADifference Estradiol 10 μg Between vs. Estradiol Treatment 90% CI forPlacebo P- Population Statistics 10 μg Placebo Means Difference¹ value²Intent-to-Treat N 24 24 — — — Least- −1.043 −1.042 −0.002 (−0.497,0.493) 0.9951 Squares Mean Mean ± SD −1.043 ± 0.928 −1.042 ± 1.08 — — —Median −1.00 −1.00 — — — Min, Max −3.00, 0.00 −3.00, 0.00 — — —¹Confidence interval for the estradiol 10 μg-Placebo from ANOVA withtreatment as a fixed effect. ²P-value for treatment comparison fromANOVA with treatment as a fixed effect.

TABLE 14 Additional Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Severity of Vaginal Atrophy SymptomsLeast-Squares TX-12-004- Mean HR vs. Statistical TX-12-004- DifferenceBetween 90% CI for Placebo Symptom Method¹ HR Placebo Treatment MeansDifference² P-value Dryness ANCOVA −0.980 −0.729 −0.251 (−0.706, 0.204)0.3597 Irritation ANCOVA −0.694 −0.514 −0.180 (−0.549, 0.189) 0.4159Pain (Sex) ANOVA −0.800 −0.500 −0.300 (−1.033, 0.433) 0.4872Pain/Burning/ ANCOVA −0.391 −0.359 −0.032 (−0.263, 0.200) 0.8185Stinging (Urination) ¹ANOVA model contained a fixed effect fortreatment. ANCOVA added baseline as a covariate to the model.²Confidence interval for the difference between TX-12-004-HR and Placebotreatment least-squares means.

With respect to the most bothersome symptoms data presented in Tables 13and 14, the period over which the data was measured is generallyconsidered insufficient to make meaningful conclusions. However, thetrends observed as part of this study suggest that the data will showimprovement of the most bothersome symptoms when data for a longer timeperiod is collected.

The absence or presence of any vaginal bleeding associated with sexualactivity was also measured as one of the most bothersome symptoms. Thedata for vaginal bleeding associated with sexual activity is reported inTable 15.

TABLE 15 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Vaginal Bleeding Associated with SexualActivity Baseline (Randomization) and Day 15 Summary of Vaginal BleedingBleeding/No Bleeding/ No Bleeding/ No Bleeding/ Bleeding BleedingBleeding No Bleeding Treatment N* (Success)² (Failure) (Failure) (NC)Estradiol 10 2 (100%) 0 0 8 10 μg Placebo 10 1 (20%)  3 1 5 P-Value for0.1429 — — — Estradiol 10 μg vs. Placebo¹ *N = Total number of patientswithin each treatment group who were sexually active at both Baselineand Day 15 and provided a response at both visits. NC = No Change - notconsidered in the statistical comparison. ¹P-value for treatmentcomparison from Fisher's Exact Test. ²Percent is based on the number ofsubjects classified as either a Success or a Failure (N = 2 forestradiol 10 μg; N = 5 for Placebo

Estradiol Level/Pharmacokinetics Data

In this study, the systemic exposure to estradiol following once dailyintravaginal administration of estradiol 10 μg for 14 days wasinvestigated. Descriptive statistics of the plasma estradiolconcentrations taken at each sampling time and the observed C_(max) andT_(max) values were recorded in Tables 16 and 17. No statisticallysignificant difference in the systemic concentration of estradiol 10 μgversus the placebo group was observed, which suggests the estradiol isnot carried into the blood stream where it will have a systemic effect.Rather, it remains in localized tissues; the effect of estradiol istherefore believed be local to the location of administration (i.e., thevagina). The lower limits of detection of the assays used to measure thepharmacokinetic data may have affected the measured the accuracy of thePK values presented. Additional PK studies were performed with moreaccurate assays in Examples 8 and 9.

For the purpose of monitoring the estradiol level during the study bloodsamples were collected at 0.0, 1.0, 3.0, and 6.0 hours relative todosing on day 1; prior to dosing on day 8; and prior to dosing on day15. Efforts were made to collect blood samples at their scheduled times.Sample collection and handling procedures for measurement of estradiolblood level was performed according to procedure approved by the sponsorand principal investigator. All baseline and post-treatment plasmaestradiol concentrations were determined using a validated bioanalytical(UPLC-MS/MS) methods. These data are shown in Tables 16 and 17.

TABLE 16 Descriptive Statistics of Estradiol Concentrations (pg/mL) atEach Sampling Time Sampling Time Pre-dose Pre-dose Treatment 0 Hour 1Hour 3 Hours 6 Hours Day 8 Day 15 Estradiol 10 μg N 24   24   24   24  24   22   Mean ± SD 20.1 ± 5.74 28.7 ± 5.89 25.7 ± 5.71 23.4 ± 7.91 21.4± 9.28 23.4 ± 8.72 Median 20.2 28.9 24.7 22.3 20.7 20.7 Min, Max 2.63,38.3 18.8, 43.9 19.3, 47.5 3.31, 52.3 2.09, 52.2 17.9, 54.7 Placebo N26   26   26   26   25   24   Mean ± SD 20.5 ± 4.29 21.0 ± 6.14 19.0 ±5.92 26.9 ± 17.36 29.9 ± 22.51 28.1 ± 16.80 Median 20.8 20.8 20.9 21.721.6 21.1 Min, Max 4.03, 29.1 3.19, 41.2 3.15, 26.9 15.1, 90.0 15.0,116.2 14.7, 81.3

TABLE 17 Descriptive Statistics of Estradiol C_(max) and T_(max) on Day1 Estradiol 10 μg Placebo C_(max) T_(max) C_(max) T_(max) N 24 24 26 26Mean ± SD 30.7 ± 7.47 2.12 ± 1.73 27.5 ± 17.26 4.00 ± 2.68 Geometric29.9 — 24.7 — Mean Median 29.8 1.00 22.1 6.00 Min, Max 19.7, 52.3 1.00,6.00 15.1, 90.0 0.00, 6.00 CV % 24.3% 81.3% 62.9% 67.1%

Assessment of Vaginal Mucosa Data

The investigators rated the vaginal mucosal appearance at day 1(pre-dose) and day 15. Vaginal color, vaginal epithelial integrity,vaginal epithelial surface thickness, and vaginal secretions wereevaluated according to the following degrees of severity: none, mild,moderate, or severe using scales 0 to 3, where 0=none, 1=mild,2=moderate, and 3=severe. Results from these investigators ratedassessments are presented in Tables 18, 19, 20, and 21.

TABLE 18 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Color) Difference Estradiol 10 μg Betweenvs. Estradiol Treatment 90% CI for Placebo P- Population Statistics 10μg Placebo Means Difference¹ value² Intent-to-Treat N 24 24 — — — Least-−0.199 −0.009 −0.191 (−0.434, 0.052) 0.1945 squares Mean Mean ± SD−0.333 ± 0.565 0.125 ± 0.741 Median 0.00 0.00 — — — Min, Max −2.00, 0.00−1.00, 2.00 — — — ¹Confidence interval for the estradiol 10 μg-Placebofrom ANCOVA with treatment as a fixed effect and baseline as acovariate. ²P-value for treatment comparison from ANCOVA with treatmentas a fixed effect and baseline as a covariate.

TABLE 19 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Epithelial Integrity) Difference Estradiol10 μg Between vs. Estradiol Treatment 90% CI for Placebo P- PopulationStatistics 10 μg Placebo Means Difference¹ value² Intent-to-Treat N 2424 — — — Least- −0.342 0.176 −0.518 (−0.726, −0.311) 0.0001 squares MeanMean ± SD −0.417 ± 0.584 0.250 ± 0.442 Median 0.00 0.00 — — — Min, Max−1.00, 1.00 0.00, 1.00 — — — ¹Confidence interval for the estradiol 10μg-Placebo from ANCOVA with treatment as a fixed effect and baseline asa covariate. ²P-value for treatment comparison from ANCOVA withtreatment as a fixed effect and baseline as a covariate.

TABLE 20 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Epithelial Surface Thickness) DifferenceEstradiol 10 μg Between vs. Estradiol Treatment 90% CI for Placebo P-Population Statistics 10 μg Placebo Means Difference¹ value²Intent-to-Treat N 24 24 — — — Least- −0.034 −0.133 0.099 (−0.024, 0.221)0.1820 squares Mean Mean ± SD −0.125 ± 0.338 −0.042 ± 0.550 — — — Median0.00 0.00 — — — Min, Max −1.00, 0.00 −1.00, 1.00 — — — ¹Confidenceinterval for the estradiol 10 μg-Placebo from ANCOVA with treatment as afixed effect and baseline as a covariate. ²P-value for treatmentcomparison from ANCOVA with treatment as a fixed effect and baseline asa covariate.

TABLE 21 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Secretions) Difference Estradiol 10 μgBetween vs. Estradiol Treatment 90% CI for Placebo P- PopulationStatistics 10 μg Placebo Means Difference¹ value² Intent-to-Treat N 2424 — — — Least- −0.643 −0.274 −0.369 (−0.661, −0.076) 0.0401 squaresMean Mean ± SD −0.792 ± 0.779 −0.125 ± 0.741 — — — Median −1.00 0.00 — —— Min, Max −2.00, 1.00 −2.00, 2.00 — — — ¹Confidence interval for theestradiol 10 μg-Placebo from ANCOVA with treatment as a fixed effect andbaseline as a covariate. ²P-value for treatment comparison from ANCOVAwith treatment as a fixed effect and baseline as a covariate.

Delivery Vehicle Disintegration Data

Assessment of capsule disintegration in the vagina (presence or absence)at Day 1 (6 hours after dosing) and Day 15. Results of this assessmentis presented in Table 22.

TABLE 22 Capsule Disintegration State in the Vagina on Day 1 and Day 15Estradiol 10 μg Placebo Day 1 Day 15 Day 1 Day 15 No evidence of 23(95.8%)  24 (100.0%)  26 (100.0%) 24 (92.3%) capsule present Evidence ofcapsule 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) present Assessment not 1(4.2%) 0 (0.0%) 0 (0.0%) 2 (7.7%) done

Serum hormone level data was collected to measure the serumconcentrations of estradiol. These data were used for screeninginclusion and were determined using standard clinical chemistry methods.

Appropriateness of Measurements

The selection of the efficacy measurements used in this study was basedon FDA's recommendations for studies of estrogen and estrogen/progestindrug products for the treatment of moderate to severe vasomotor symptomsassociated with the menopause and moderate to severe symptoms of vulvarand vaginal atrophy associated with the menopause (Food and DrugAdministration, Guidance for Industry, Estrogen and Estrogen/ProgestinDrug Products to Treat Vasomotor Symptoms and Vulvar and Vaginal AtrophySymptoms—Recommendations for Clinical Evaluation. January 2003, herebyincorporated by reference).

Standard clinical, laboratory, and statistical procedures were utilizedin the trial. All clinical laboratory procedures were generally acceptedand met quality standards.

Statistical Methods:

Efficacy:

Analysis of variance (ANOVA) was used to evaluate the change frombaseline differences between the subjects receiving estradiol 10 μg andplacebo capsules for all efficacy endpoints, except for vaginalbleeding, to estimate the effect size and variability of the effect. Insome cases, for example, for some vaginal atrophy symptoms, the changefrom baseline (post dose response) was correlated with the baselinevalue (p<0.05), so baseline was included as a covariate to adjust forthis correlation (Analysis of Covariance, ANCOVA). The 90% confidenceintervals on the differences between estradiol 10 μg and placeboendpoint means were determined to evaluate the effect size. The changefrom baseline in vaginal bleeding associated with sexual activity wasevaluated in terms of the proportion of subjects who had treatmentsuccess or failure. Any subject reporting bleeding at baseline who didnot report bleeding at Day 15 was considered to have been successfullytreated. Any subject reporting bleeding at day 15 was considered atreatment failure, regardless of whether they reported baseline bleedingor not. Subjects reporting no bleeding at both baseline and day 15 wereclassified as no-change and were excluded from the statisticalevaluation. The difference in the proportion of subjects with successbetween the two treatment groups was statistically evaluated usingFisher's Exact Test. Results of this difference in proportion arepresented in Table 10.

Measurements of Treatment Compliance

Subjects were required to complete a diary in order to record treatmentcompliance. Diaries were reviewed for treatment compliance at day 8 andday 15 visits. A total of 45 subjects (21 subjects in the estradiol 10μg group and 24 subjects in the placebo group) were 100% compliant withthe treatment regimen.

Due to the investigative nature of the study, no adjustments were madefor multiplicity of endpoints.

Safety:

The frequency and severity of all adverse events were summarizeddescriptively by treatment group.

Results: All forty eight (48) subjects who completed the study wereincluded in the primary efficacy analyses. The results of efficacyanalyses are presented throughout Tables 5, 6, and 7.

Conclusions

Efficacy

The two-week treatment with pharmaceutical composition 10 μg led to astatistically significant greater mean decrease in percent of parabasalcells than did placebo treatment (54% vs. 5%, p<0.0001), as illustratedin Table 6. At the same time, a significantly greater mean increase inthe percent of superficial cells was observed with the pharmaceuticalcomposition (35%) than with the placebo capsules (9%), with thedifference being highly statistically significant (p=0.0002), asillustrated in Table 7. The difference in pH reduction between thepharmaceutical composition (0.97 units) compared to that for the placebo(0.34 units) was only slightly greater than 0.5 units, but thedifference was detected as statistically significant (p=0.0002), asillustrated in Table 9.

While the decrease in severity of the most bothersome symptom wasessentially the same (˜1 unit) for both pharmaceutical composition andplacebo, the reductions in the severity of the individual symptoms ofvaginal dryness, irritation and pain during sexual activity were allmarginally better for the active treatment than for the placebotreatment. None of the differences between the two treatments, all ofwhich were <0.3 units, were detected as statistically significant. Therewas no difference between the two treatments in regard to reduction ofpain/burning/stinging during urination (˜0.4 unit reduction). The lengthof the study was not long enough to show a separation between the mostbothersome symptoms in the pharmaceutical composition and placebo.However, the trends of most bothersome symptoms suggest that with asuitable period of time, significantly significant differences betweenthe two treatments would be observed.

The two-week treatment with estradiol 10 μg capsules showed nostatistically detectable difference in regard to reduction of severityfrom baseline according to the investigator's assessment of vaginalcolor or vaginal epithelial surface thickness. Pharmaceuticalcomposition capsules did demonstrate a statistically significant greaterreduction than did placebo in severity of atrophic effects on vaginalepithelial integrity (˜0.34 vs. 0.18, p=0.0001) and vaginal secretions(−0.64 vs. −0.27, p=0.0401).

Descriptive statistical analyses (mean, median, geometric mean, standarddeviation, CV, minimum and maximum, C_(max), and T_(max)) were conductedon the estradiol concentrations at each sampling time, the peakconcentration on day 1 and the time of peak concentration. Results fromthis assessment are presented in Tables 16 and 17.

A pharmaceutical composition comprising estradiol 10 μg outperformedplacebo treatment in regard to improvement in the Maturation Index,reduction in vaginal pH, reduction in the atrophic effects on epithelialintegrity and vaginal secretions. The lack of statistical significancebetween the two treatments in regard to reduction of severity for themost bothersome symptom, and the individual vaginal atrophy symptoms ofdryness, irritation, pain associated with sexual activity, andpain/burning/stinging during urination, is not unexpected given thesmall number of subjects in the study and the short duration of therapy.Too few subjects in the study had vaginal bleeding associated withsexual activity to permit any meaningful evaluation of this vaginalatrophy symptom.

Of the 48 subjects enrolled in the study, 45 subjects were 100%compliant with the treatment regimen. Of the remaining three subjects,one removed herself from the study due to personal reasons and the othertwo subjects each missed one dose due to an adverse event.

Safety

Although the Day 1 mean plasma estradiol peak concentration for thepharmaceutical composition was somewhat higher than that for the Placebo(ratio of geometric means=1.21: Test Product (estradiol 10 μg)21%>Placebo), no statistically significant difference was determined.However, the assay methods were questionable, resulting in questionablePK data. Additional PK studies were performed in Examples 8 and 9.

There were no serious adverse events in the study.

Overall, the pharmaceutical composition comprising estradiol 10 μg waswell tolerated when administered intravaginally in once daily regimenfor 14 days.

Example 8 PK Study (25 μg Formulation)

A PK study was undertaken to compare the 25 μg formulation disclosedherein (Pharmaceutical Composition 3) to the RLD. The results of the PKstudy for estradiol are summarized in Table 23. The p values for thesedata demonstrate statistical significance, as shown in Table 24.

TABLE 23 Statistical Summary of the Comparative Bioavailability Data forUnscaled Average BE studies of Estradiol, Least Square Geometric Meansof Estradiol, Ratio of Means and 90% Confidence Intervals, Fasting/FedBioequivalence Study (Study No.: ESTR-1K-500-12), Dose 25 μg estradiolParameter Test N RLD N Ratio (%) 90% C.I. C_(max) 23.0839 36 42.7024 3654.06 44.18-66.14 (pg/mL) AUC₀₋₂₄ 89.2093 36 292.0606 36 30.5423.72-39.34 (pg · hr/mL)

TABLE 24 P-values for Table 23 P-Value Effect C_(max) AUC₀₋₂₄ Treatment<.0001 <.0001 Sequence 0.4478 0.5124 Period 0.4104 0.7221

As illustrated in Table 23, baseline adjusted PK data illustrates thatthe formulations disclosed herein unexpectedly show a 54% decrease inC_(max) and a 31% decrease in the AUC relative to the RLD. This resultis desirable because the estradiol is intended only for localabsorption. These data suggest a decrease in the circulating levels ofestradiol relative to the RLD. Moreover, it is noteworthy to point outthat the C_(max) and AUC levels of estradiol relative to placebo are notstatistically differentiable, which suggests that the formulationsdisclosed herein have a negligible systemic effect. As shown in Table24, there was no significant difference between the test and referenceproducts due to sequence and period effects. However, there was asignificant difference due to treatment effect for both C_(max) and AUC.

Pharmacokinetics for circulating total estrone, a metabolite ofestradiol, is show in Table 25. These data show that the totalcirculating estrone for the formulations disclosed herein resulted in a55% decrease in the C_(max) for circulating estrone, and a 70% decreasein the AUC for circulating estrone.

TABLE 25 Statistical Summary of the Comparative Bioavailability Data forUnscaled Average BE studies of Estrone, Least Square Geometric Means,Ratio of Means and 90% Confidence Intervals, Fasting/Fed BioequivalenceStudy (Study No.: ESTR-1K-500-12), Dose 25 μg estradiol Parameter Test NRLD N Ratio (%) 90% C.I. C_(max) 10.7928 36 23.5794 36 45.77 32.95 to63.59 (pg/mL) AUC₀₋₂₄ 51.2491 36 165.4664 36 30.97 19.8-48.45 (pg ·hr/mL)

TABLE 26 P-values for Table 25 P-Value Effect C_(max) AUC₀₋₂₄ Treatment0.0002 <.0001 Sequence 0.1524 0.0464 Period 0.0719 0.0118

There was a significant difference between test and reference productsdue to treatment effect whereas there was no significant difference dueto sequence and period effects for C_(max). For AUC, there was asignificant difference between test and reference products due totreatment, sequence, and period effects.

PK for circulating total estrone sulfate is shown in Table 27. Thesedata show that the total circulating estrone sulfate for thepharmaceutical compositions disclosed herein resulted in a 33% decreasein the C_(max) and a 42% decrease in the AUC for circulating estronesulfate.

TABLE 27 Statistical Summary of the Comparative Bioavailability Data forUnscaled Average BE studies of Estrone Sulfate, Least Square GeometricMeans of Estrone Sulfate, Ratio of Means and 90% Confidence Intervals,Fasting/Fed Bioequivalence Study (Study No.: ESTR-1K-500-12), Dose 25 μgestradiol Parameter Test N RLD N Ratio (%) 90% C.I. C_(max) 490.0449 36730.5605 36 67.08 53.84-83.57 (pg/mL) AUC₀₋₂₄ 4232.9914 36 7323.0827 3657.80 43.23-77.29 (pg · hr/mL)

TABLE 28 P-values for Table 27 P-Value Effect C_(max) AUC₀₋₂₄ Treatment0.0042 0.0031 Sequence 0.5035 0.9091 Period 0.1879 0.8804

There was a significant difference between test and reference productsdue to treatment effect whereas there was no significant difference duesequence and period effects for both C_(max) and AUC.

Example 9 PK Study (10 μg Formulation)

A PK study was undertaken to compare the 10 μg formulation disclosedherein (Pharmaceutical Composition 2) to the RLD. The results of the PKstudy for estradiol are summarized in Table 29-40, and FIGS. 9-14.

A PK study was undertaken to compare pharmaceutical compositionsdisclosed herein having 10 μg of estradiol to the RLD. The results ofthe PK study for estradiol are summarized in Tables 29-34, whichdemonstrate that the pharmaceutical compositions disclosed herein moreeffectively prevented systemic absorption of the estradiol. Table 35shows that the pharmaceutical compositions disclosed herein had a 28%improvement over the RLD for systemic blood concentration C_(max) and72% AUC improvement over the RLD.

TABLE 29 Summary of Pharmacokinetic Parameters of Test product (T) ofEstradiol - Baseline adjusted (N = 34) Arithmetic Pharmaco- Mean ±Coeffi- kinetic Standard cient of Me- Mini- Maxi- Parameter DeviationVariation dian mum mum C_(max) 15.7176 ± 50.3761 13.9000 6.5000 49.6000(pg/mL) 7.9179 AUC₀₋₂₄ 53.0100 ± 36.9041 49.9750 24.3000 95.1500 (pg ·hr/mL) 19.5629 t_(max) (hr) 1.98 ± 65.34 2.00 1.00 8.05 1.29

TABLE 30 Summary of Pharmacokinetic Parameters of Reference product (R)of Estradiol - Baseline adjusted (N = 34) Arithmetic Pharmaco- Mean ±Coeffi- kinetic Standard cient of Me- Mini- Maxi- Parameter DeviationVariation dian mum mum C_(max) 24.1882 ± 49.2877 24.1500 1.0000 55.3000(pg/mL) 11.9218 AUC₀₋₂₄ 163.8586 ± 43.9960 158.0375 2.0000 304.8500 (pg· hr/mL) 72.0913 t_(max) (hr) 10.53 ± 52.94 8.06 2.00 24.00 5.58

TABLE 31 Geometric Mean of Test Product (T) and Reference product (R) ofEstradiol - Baseline adjusted (N = 34) Geometric Mean PharmacokineticParameter Test Product (T) Reference Product (R) C_(max) (pg/mL) 14.377420.3837 AUC₀₋₂₄ (pg · hr/mL) 49.6231 132.9218 t_(max) (hr) 1.75 9.28

TABLE 32 Statistical Results of Test product (T) versus Referenceproduct (R) for Estradiol - Baseline adjusted (N = 34) Geometric LeastSquare Mean Test Reference Intra 90% Pharmacokinetic Product ProductSubject T/R Confidence Parameter (T) (R) CV % Ratio % Interval C_(max)(pg/mL) 14.4490 20.1980 60.68 71.54* 56.82-90.08 AUC₀₋₂₄ 49.7310131.0400 70.64 37.95* 29.21-49.31 (pg · hr/mL) *Comparison was detectedas statistically significant by ANOVA (α = 0.05).

The PK data for total estrone likewise demonstrated reduced systemicexposure when compared to the RLD. Table 33 shows the pharmaceuticalcompositions disclosed herein reduced systemic exposure by 25% forC_(max) and 49% for AUC.

TABLE 33 Summary of Pharmacokinetic Parameters of Test product (T) ofEstrone - Baseline adjusted (N = 33) Arithmetic Pharmaco- Mean ± Coeffi-kinetic Standard cient of Me- Mini- Maxi- Parameter Deviation Variationdian mum mum C_(max) 6.8485 ± 96.1149 5.4000 1.3000 36.3000 (pg/mL)6.5824 AUC₀₋₂₄ 34.7051 ± 80.5476 30.8500 3.3500 116.7500 (pg · hr/mL)27.9541 t_(max) (hr) 9.12 ± 96.80 4.00 1.00 24.00 8.83

TABLE 34 Summary of Pharmacokinetic Parameters of Reference product (R)of Estrone - Baseline adjusted (N = 33) Arithmetic Pharmaco- Mean ±Coeffi- kinetic Standard cient of Me- Mini- Maxi- Parameter DeviationVariation dian mum mum C_(max) 8.8333 ± 80.9086 6.7000 2.7000 30.3000(pg/mL) 7.1469 AUC₀₋₂₄ 63.0042 ± 73.8814 51.2800 8.8000 214.0000 (pg ·hr/mL) 46.5484 t_(max) (hr) 11.16 ± 64.95 10.00 4.00 24.00 7.24

TABLE 35 Geometric Mean of Test Product (T) and Reference product (R) ofEstrone - Baseline adjusted (N = 33) Geometric Mean PharmacokineticParameter Test Product (T) Reference Product (R) C_(max) (pg/mL) 5.15076.9773 AUC₀₋₂₄ (pg · hr/mL) 24.2426 48.2377 t_(max) (hr) 5.87 9.07

TABLE 36 Statistical Results of Test product (T) versus Referenceproduct (R) for Estrone - Baseline adjusted (N = 33) Geometric LeastSquare Mean Test Reference Intra 90% Pharmacokinetic Product ProductSubject T/R Confidence Parameter (T) (R) CV % Ratio % Interval C_(max)(pg/mL) 5.1620 6.9280 47.59 74.50* 61.69-89.97 AUC₀₋₂₄ 24.1960 47.902073.66 50.51* 38.37-66.50 (pg · hr/mL) *Comparison was detected asstatistically significant by ANOVA (α = 0.05).

The PK data for estrone sulfate likewise demonstrated reduced systemicexposure when compared to the RLD. Table 37 shows the pharmaceuticalcompositions disclosed herein reduced systemic exposure by 25% forC_(max) and 42% for AUC.

TABLE 37 Summary of Pharmacokinetic Parameters of Test product (T) ofEstrone Sulfate - Baseline adjusted (N = 24) Arithmetic Mean ±Pharmacokinetic Standard Coefficient Parameter Deviation of VariationMedian Minimum Maximum C_(max) (ng/mL) 13.9042 ± 7.0402  50.6339 11.15001.3000 39.0000 AUC₀₋₂₄ (ng · hr/mL) 97.9953 ± 80.8861 82.5408 76.27505.1025 338.0000 t_(max) (hr) 6.33 ± 4.56 71.93 4.00 4.00 24.00

TABLE 38 Summary of Pharmacokinetic Parameters of Reference product (R)of Estrone Sulfate - Baseline adjusted (N = 24) Arithmetic Mean ±Pharmacokinetic Standard Coefficient Parameter Deviation of VariationMedian Minimum Maximum C_(max) (ng/mL) 19.2542 ± 11.3633 59.0173 15.20007.0000 53.7000 AUC₀₋₂₄ (ng · hr/mL) 177.6208 ± 166.2408 93.5931 124.000020.0000 683.0500 t_(max) (hr) 10.33 ± 5.58  54.05 10.00 2.00 24.00

TABLE 39 Geometric Mean of Test Product (T) and Reference product (R) ofEstrone Sulfate - Baseline adjusted (N = 24) Geometric MeanPharmacokinetic Parameter Test Product (T) Reference Product (R) C_(max)(ng/mL) 12.1579 16.8587 AUC₀₋₂₄ (ng · hr/mL) 66.5996 121.5597 t_(max)(hr) 5.49 8.83

TABLE 40 Statistical Results of Test product (T) versus Referenceproduct (R) for Estrone Sulfate - Baseline adjusted (N = 24) GeometricLeast Square Mean Test Reference Intra 90% Pharmacokinetic ProductProduct Subject T/R Confidence Parameter (T) (R) CV % Ratio % IntervalC_(max) (ng/mL) 12.3350 16.5470 48.02 74.55* 59.43-93.51 AUC₀₋₂₄ 68.5260118.4170 73.87 57.87* 41.68-80.35 (ng · hr/mL) *Comparison was detectedas statistically significant by ANOVA (α = 0.05).

Example 10 Randomized, Double-Blind, Placebo-Controlled MulticenterStudy of Estradiol Vaginal Softgel Capsules for Treatment of VVAInvestigational Plan

The study was a randomized, double-blind, placebo-controlled multicenterstudy design. Postmenopausal subjects who meet the study entry criteriawill be randomized in a 1:1:1:1 ratio to receive Estradiol VaginalSoftgel Capsule 4 μg, Estradiol Vaginal Softgel Capsule 10 μg, EstradiolVaginal Softgel Capsule 25 μg, or matching placebo. Subjects will beasked to self-assess the symptoms of vulvar or vaginal atrophy includingvaginal pain associated with sexual activity, vaginal dryness, vulvar orvaginal itching or irritation by completing the VVA symptomself-assessment questionnaire and identification of her MBS at screeningvisit 1A to determine eligibility for the study. The VVA symptomSelf-Assessment Questionnaire, vaginal cytology, vaginal pH, and vaginalmucosa will be assessed at screening visit 1B. These assessments willdetermine continued eligibility and will be used as the baselineassessments for the study. Randomized subjects will then complete theQuestionnaire during visits 3, 4, 5, and 6.

The primary efficacy endpoints for the study included: (A) change frombaseline to week 12 in the percentage of vaginal superficial cells (byvaginal cytologic smear) compared to placebo; (B) change from baselineto week 12 in the percentage of vaginal parabasal cells (by vaginalcytologic smear) compared to placebo; (C) change from baseline at week12 in vaginal pH as compared to placebo; and (D) change from baseline toweek 12 on the severity of the MBS of dyspareunia (vaginal painassociated with sexual activity) associated with VVA as compared toplacebo.

The secondary efficacy endpoints for the study included: (E) change frombaseline to weeks 2, 6, and 8 in the percentage of vaginal superficialcells (by vaginal cytologic smear) compared to placebo; (F) change frombaseline to weeks 2, 6, and 8 in the percentage of vaginal parabasalcells (by vaginal cytologic smear) compared to placebo; (G) change frombaseline to weeks 2, 6, and 8 in vaginal pH as compared to placebo; (H)change from baseline to weeks 2, 6, and 8 on the severity of the MBS ofdyspareunia (vaginal pain associated with sexual activity) associatedwith VVA as compared to placebo; (I) change from baseline to weeks 2, 6,8, and 12 on the severity of vaginal dryness and vulvar or vaginalitching or irritation associated with VVA as compared to placebo; (J)change in visual evaluation of the vaginal mucosa from baseline to weeks2, 6, 8, and 12 compared to placebo; (K) assessment of standard PKparameters as defined in the SAP for serum estradiol, estrone, andestrone conjugates at Screening Visit 1A, days 1, 14, and 84 oftreatment in a subset of subjects (PK substudy) utilizing baselinecorrected and uncorrected values [as outlined in the StatisticalAnalysis Plan (SAP)]; and (L) change from baseline in the Female SexualFunction Index (FSFI) at week 12 compared to placebo.

The safety endpoints for the study included: (1) Adverse events; (2)Vital signs; (3) Physical examination findings; (4) Gynecologicalexamination findings; (5) Clinical laboratory tests; (6) Pap smears; and(7) Endometrial biopsy.

Approximately 100 sites in the United States and Canada screenedapproximately 1500 subjects to randomize 747 subjects in this study(modified intent to treatment population, or all subjects who have takenat least one dose of the pharmaceutical compositions disclosed herein),with a target of 175 subjects randomized to each treatment group (175 ineach active treatment group and 175 in the placebo group to complete 560subjects). Actual subjects enrolled are 186 subjects in the 4 μgformulation group, 188 subjects in the 10 μg formulation group, 186subjects in the 25 μg formulation group, and 187 subjects in the placebogroup, for a total of 747 subjects in the study. Within each treatmentgroup, 15 subjects also participated in a PK substudy. Subjects wereassigned to one of four treatment groups: (1) 4 μg formulation; (2) 10μg formulation; (3) 25 μg formulation; and (4) placebo.

Most subjects participated in the study for 20-22 weeks. This included a6 to 8 week screening period (6 weeks for subjects without an intactuterus and 8 weeks for subjects with an intact uterus), 12 weeks on theinvestigational product, and a follow-up period of approximately 15 daysafter the last dose of investigational product. Some subjects'involvement lasted up to 30 weeks when an 8-week wash-out period wasnecessary. Subjects who withdrew from the study were not replacedregardless of the reason for withdrawal.

The study schematic diagram shown in FIG. 9. There were two treatmentperiods; once daily intravaginal administration of one of the listedinvestigational products for 2 weeks, followed by a twice weeklyintravaginal administration for 10 weeks.

The subject inclusion criteria included: (1) postmenopausal femalesubjects between the ages of 40 and 75 years (at the time ofrandomization) with at least: 12 months of spontaneous amenorrhea (women<55 years of age with history of hysterectomy without bilateraloophorectomy prior to natural menopause must have follicle stimulatinghormone (FSH) levels >40 mIU/mL); or 6 months of spontaneous amenorrheawith follicle stimulating hormone (FSH) levels >40 m1U/mL; or At least 6weeks postsurgical bilateral oophorectomy.

The subject inclusion criteria also included: (2) ≤5% superficial cellson vaginal cytological smear; (3) Vaginal pH>5.0; (4) Moderate to severesymptom of vaginal pain associated with sexual activity considered themost bothersome vaginal symptom by the subject at screening visit IA;(5) Moderate to severe symptom of vaginal pain associated with sexualactivity at screening visit 1B; (6) Onset of moderate to severedyspareunia in the postmenopausal years; (7) Subjects were sexuallyactive (i.e., had sexual activity with vaginal penetration withinapproximately 1 month of screening visit 1A); and (8) Subjectsanticipated having sexual activity (with vaginal penetration) during theconduct of the trial

For subjects with an intact uterus, the subject inclusion criteria alsoincluded: (9) subjects had an acceptable result from an evaluablescreening endometrial biopsy. The endometrial biopsy reports by the twocentral pathologists at screening specified one of the following:proliferative endometrium; weakly proliferative endometrium; disorderedproliferative pattern; secretory endometrium; endometrial tissue other(i.e., benign, inactive, or atrophic fragments of endometrialepithelium, glands, stroma, etc.); endometrial tissue insufficient fordiagnosis; no endometrium identified; no tissue identified; endometrialhyperplasia; endometrial malignancy; or other findings (endometrialpolyp not present, benign endometrial polyp, or other endometrialpolyp). Identification of sufficient tissue to evaluate the biopsy by atleast one pathologist was required.

For subjects with a Body Mass Index (BMI) less than or equal to 38kg/m², the subject inclusion criteria also included: (10) BMI valueswere rounded to the nearest integer (ex. 32.4 rounds down to 32, while26.5 rounds up to 27).

In general, the inclusion criteria also included: (11) in the opinion ofthe investigator, the subject was believed likely to comply with theprotocol and complete the study.

The exclusion criteria included: (1) use of oral estrogen-, progestin-,androgen-, or SERM-containing drug products within 8 weeks beforescreening visit 1A (entry of washout was permitted); use of transdermalhormone products within 4 weeks before screening visit 1A (entry ofwashout was permitted); use of vaginal hormone products (rings, creams,gels) within 4 weeks before screening visit 1A (entry of washout waspermitted); use of intrauterine progestins within 8 weeks beforescreening visit 1A (entry of washout was permitted); use of progestinimplants/injectables or estrogen pellets/injectables within 6 monthsbefore screening visit 1A (entry of washout was not permitted); or useof vaginal lubricants or moisturizers within 7 days before the screeningvisit 1B vaginal pH assessment.

The exclusion criteria also included: (2) a history or active presenceof clinically important medical disease that might confound the study orbe detrimental to the subject, including, for example: hypersensitivityto estrogens; endometrial hyperplasia; undiagnosed vaginal bleeding; ahistory of a chronic liver or kidney dysfunction/disorder (e.g.,Hepatitis C or chronic renal failure); thrombophlebitis, thrombosis, orthromboembolic disorders; cerebrovascular accident, stroke, or transientischemic attack; myocardial infarction or ischemic heart disease;malignancy or treatment for malignancy, within the previous 5 years,with the exception of basal cell carcinoma of the skin or squamous cellcarcinoma of the skin (a history of estrogen dependent neoplasia, breastcancer, melanoma, or any gynecologic cancer, at any time, excluded thesubject); and endocrine disease (except for controlled hypothyroidism orcontrolled non-insulin dependent diabetes mellitus).

The exclusion criteria also included: (3) recent history of knownalcohol or drug abuse; (4) history of sexual abuse or spousal abuse thatwas likely to interfere with the subject's assessment of vaginal painwith sexual activity; (5) current history of heavy smoking (more than 15cigarettes per day) or use of e-cigarettes; (6) use of an intrauterinedevice within 12 weeks before screening visit 1A; (7) use of aninvestigational drug within 60 days before screening visit 1A; (8) anyclinically important abnormalities on screening physical exam,assessments, electrocardiogram (ECG), or laboratory tests; (9) knownpregnancy or a positive urine pregnancy test; and (10) current use ofmarijuana.

In this study, if a subject discontinued or was withdrawn, the subjectwas not replaced. At the time of consent, each subject was given aunique subject number that identified their clinical site and sequentialnumber. In addition to the assigned subject number, subject initialswere used for identification. The clinical trial was performed incompliance with standard operating procedures as well as regulations setforth by FDA, ICH E6 (R1) guidelines, and other relevant regulatoryauthorities. Compliance was achieved through clinical trial-specificaudits of clinical sites and database review.

Statistical Methods

Efficacy. The primary objective of the trial was to assess the efficacyof estradiol vaginal softgel capsules (4 μg, 10 μg, and 25 μg) whencompared to placebo on vaginal superficial cells, vaginal parabasalcells, vaginal pH, and on the symptom of moderate to severe dyspareunia(vaginal pain associated with sexual activity) as the MBS at week 12. Toaccount for the multiple comparisons of testing placebo to each of thethree doses of estradiol (4 μg, 10 μg, and 25 μg) and the multipletesting of the four co-primary endpoints, a closed procedure wasperformed (see, Edwards D, Madsen J. “Constructing multiple proceduresfor partially ordered hypothesis sets.” Stat Med 2007:26-5116-24,incorporated by reference herein).

Determination of Sample Size. The sample size needed per dose vs.placebo for each test of hypothesis in the modified intent-to-treat(MITT) population to achieve a given power was calculated usingreference data from other studies (see, Bachman, G., et al. “Efficacyand safety of low-dose regimens of conjugated estrogens creamadministered vaginally.” Menopause, 2009. 16(4): p. 719-27; Simon, J.,et al. “Effective Treatment of Vaginal Atrophy With an Ultra-Low-DoseEstradiol Vaginal Tablet.” Obstetrics & Gynecology, 2008. 112(5):p.1053-60; FDA Medical Officer's Review of Vagifem [NDA 20-908, Mar. 25,1999, Table 6, p 12.], each incorporated by reference herein). Table 41below provides the effect sizes, power, and sample size determinationsfor each of the primary endpoints. In general, subjects in the study metall inclusion/exclusion criteria and had moderate to severe dyspareuniaas their most bothersome symptom of VVA. Based on the power analysis andthe design considerations, approximately 175 subjects per treatment armwere enrolled.

TABLE 41 Power Analysis and Sample Size Determinations Four PrimaryEndpoints in a Closed Procedure Mean Change from Baseline to Week 12Compared to Placebo (MMRM) Power (One-way ANOVA, Alpha = 0.005,one-tailed) Power Based Upon Primary Endpoint Effect Size (%)* N = 140per group per MITT % Parabasal Cells 150.3% >0.999 % Superficial115.3% >0.999 Cells Vaginal pH  77.4% >0.999 Severity of 30.0%, 41.2%,70.5% 0.50, 0.80, >0.999 Dyspareunia** *Range from 30% (Vagifem 10 μg;see, Simon 2008, supra), 41.2% (Vagifem 25 μg; see, FDA 1999, supra),70.5% (Premarin cream 2/week; see, Bachman 2009, supra) **Effect Size iscalculated for all primary endpoints as 100% times difference (treatedminus placebo) in mean changes at week 12 from baseline.

All subjects who were randomly assigned and had at least 1 dose ofinvestigational product formed the intent-to-treat (ITT) population. TheModified intent-to-treat (MITT) population was defined as all ITTsubjects with a baseline and at least one follow-up value for each ofthe primary endpoints, each subject having taken at least one dose ofinvestigational product, and was the primary efficacy population. Theefficacy-evaluable (EE) population was defined as all MITT subjects whocompleted the clinical trial, were at least 80% compliant withinvestigational product, had measurements for all primary efficacyendpoints, and were deemed to be protocol compliant, with no significantprotocol violations. The safety population included all ITT subjects.

The primary efficacy analyses were conducted on the MITT subjects withsupportive efficacy analyses conducted on the EE population. Foranalysis purposes, subjects were required to complete all visits, up toand including Visit 6 (week 12), to be considered as having completedthe study.

Analysis of Efficacy Endpoints. For all numerically continuous efficacyendpoints, which included the four primary endpoints (mean change frombaseline to week 12), active treatment group means were compared toplacebo using an ANCOVA adjusting for the baseline level.

Primary and secondary efficacy endpoints were measured at baseline andat 2, 6, 8, and 12 weeks. The analysis examined change from baseline.Therefore, ANCOVAs were based on a repeated measures mixed effects model(MMRM) where the random effect was subject and the two fixed effectswere treatment group and visit (2, 6, 8, and 12 weeks). Baselinemeasures and age were used as covariates. ANCOVAs were therefore notcalculated independently for each study collection period. The analysesstarted with the full model but, interaction terms for visit (week 2, 6,8, and 12) with treatment only remained where statistically significant(p<0.05).

The following three pair-wise comparisons were performed using theappropriate ANCOVA contrast for week 12 (primary) and weeks 2, 6, and 8(secondary) changes from baseline: (1) active treatment, high dose groupvs placebo; (2) active treatment, middle dose group vs placebo; and (3)active treatment, low dose group vs placebo.

Safety outcome measures. Adverse events, vital signs, physicalexamination findings, gynecological examination findings, clinicallaboratory tests, pap smears, and endometrial biopsy were the safetyparameters. Adverse events and SAEs were summarized for each treatmentgroup and overall for all active treatment groups with the proportion ofsubjects reporting each event. Actual values and change from baseline invital signs, and all laboratory test parameters were summarized for eachtreatment group and overall for all active treatment groups withdescriptive statistics at each assessment obtained.

Endometrial Biopsy Assessment. Three independent pathologists withexpertise in gynecologic pathology, blinded to treatment and to eachother's readings, determined the diagnosis for endometrial biopsy slidesduring the conduct of the study. All visit 6, early termination, andon-treatment unscheduled endometrial biopsies were centrally read bythree of the pathologists. Each pathologist's report was classified intoone of the following three categories: category 1: not hyperplasia/notmalignancy—includes proliferative endometrium, weakly proliferativeendometrium, disordered proliferative pattern, secretory endometrium,endometrial tissue other (i.e., benign, inactive or atrophic fragmentsof endometrial epithelium, glands, stroma, etc.), endometrial tissueinsufficient for diagnosis, no endometrium identified, no tissueidentified, other; category 2: hyperplasia—includes simple hyperplasiawith or without atypia and complex hyperplasia with or without atypia;category 3: malignancy—endometrial malignancy.

The final diagnosis was based on agreement of two of the three reads.Consensus was reached when two of the three pathologist readers agreedon any of the above categories. For example, any 2 subcategories of “nothyperplasia/not malignancy” were classified as “Category 1: nothyperplasia/not malignancy.” If all three readings were disparate (i.e.,each fell into a different category—category 1, 2, or 3), the finaldiagnosis was based on the most severe of the three readings.

The analysis population for endometrium hyperplasia was the endometrialhyperplasia (EH) population. An EH subject at week 12 was one who wasrandomly assigned and took at least 1 dose of investigational product,with no exclusionary protocol violation (as detailed at the StatisticalAnalysis Plan), and had a pretreatment endometrial biopsy and a biopsyon therapy.

Treatment of Subjects

The study used a double-blind design. Investigational product wassupplied as 3 doses of Estradiol Vaginal Softgel Capsules (4 μg, 10 μg,and 25 μg) and matching placebo capsules. All subjects manually insertedone capsule into the vaginal cavity daily for 14 days (2 weeks) followedby twice weekly for 10 weeks according to one of the following treatmentarms:

TABLE 42 Treatment Arms and Administration Regimen Capsules CapsulesTreatment 1 1 capsule daily of 4 μg vaginal 1 capsule twice softgel for2 weeks weekly of 4 μg vaginal softgel for 10 weeks Treatment 2 1capsule daily of 10 μg vaginal 1 capsule twice softgel for 2 weeksweekly of 10 μg vaginal softgel for 10 weeks Treatment 3 1 capsule dailyof 25 μg vaginal 1 capsule twice weekly of 25 μg vaginal softgel for 2weeks softgel for 10 weeks Treatment 4 1 capsule daily of placebovaginal 1 capsule twice weekly softgel for 2 weeks of placebo vaginalsoftgel for 10 weeks

Investigational product was dispensed to all eligible subjects at visit2. Each subject was provided a total of 30 soft gel capsules ofinvestigational product in a labeled bottle, allowing for extra capsulesfor accidental loss or damage. A second bottle was dispensed at Visit 5.Each subject was trained by the clinical site to self-administerintravaginally one capsule daily at approximately the same hour for 2weeks (14 days). The drug administration instructions included: “Removevaginal capsule from the bottle; find a position most comfortable foryou; insert the capsule with the smaller end up into vaginal canal forabout 2 inches.” Starting on Day 15, each subject administered 1 capsuletwice weekly for the remaining 10 weeks. Twice weekly dosing wasapproximately 3-4 days apart, and generally did not exceed more thantwice in a seven day period. For example, if the Day 15 dose wasinserted on Sunday, the next dose was inserted on Wednesday or Thursday.At randomization visit 2 (day 1), subjects received their first dose ofinvestigational product at the clinical facility under the supervisionof the study personnel.

The investigational estradiol vaginal softgel drug products used in thestudy are pear-shaped, opaque, light pink softgel capsules. The capsulescontain the solubilized estradiol pharmaceutical compositions disclosedherein as Pharmaceutical Compositions 4-7. When the softgel capsulescome in contact with the vaginal mucosa, the soft gelatin capsulereleases the pharmaceutical composition, into the vagina. Inembodiments, the soft gelatin capsule completely dissolves.

The placebo used in the study contained the excipients in theinvestigational estradiol vaginal softgel capsule without the estradiol(see, e.g., Pharmaceutical Composition 7). The packaging of theinvestigational products and placebo were identical to maintain adequateblinding of investigators. Neither the subject nor the investigator wasable to identify the treatment from the packaging or label of theinvestigational products.

A subject was required to use at least 80% of the investigationalproduct to be considered compliant with investigational medicationadministration. Capsule count and diary cards were be used to determinesubject compliance at each study visit. Subjects were randomly assignedin a 1:1:1:1 ratio to receive Estradiol Vaginal Softgel Capsule 4 μg(Pharmaceutical Composition 4), Estradiol Vaginal Softgel Capsule 10 μg(Pharmaceutical Composition 5), Estradiol Vaginal Softgel Capsule 25 μg(Pharmaceutical Composition 6), or placebo (Pharmaceutical Composition7).

Concomitant medications/treatments were used to treat chronic orintercurrent medical conditions at the discretion of the investigator.The following medications were prohibited for the duration of the study:investigational drugs other than the investigational Estradiol VaginalSoftgel Capsule; estrogen-, progestin-, androgen (i.e., DHEA) orSERM-containing medications other than the investigational product;medications, remedies, and supplements known to treat vulvar/vaginalatrophy; vaginal lubricants and moisturizers (e.g., Replens) bediscontinued 7 days prior to Visit 1B vaginal pH assessment; and allmedications excluded before the study.

Efficacy Assessments

Vaginal cytological smears were collected from the lateral vaginal wallsaccording to standard procedures and sent to a central laboratory toevaluate vaginal cytology. The percentage of superficial, parabasal, andintermediate cells was determined. All on-therapy/early terminationvaginal cytology results were blinded to the Sponsor, Investigators, andsubjects.

Vaginal pH was determined at screening Visit 1B and visits 3, 4, 5, and6/end of treatment. Subjects were not allowed to use vaginal lubricantsor moisturizers within 7 days of the screening vaginal pH assessment orat any time afterwards during the study. The subjects were advised notto have sexual intercourse and to refrain from using vaginal douchingwithin 24 hours prior to the measurement for all scheduled vaginal pHassessments. After insertion of an unlubricated speculum, a pH indicatorstrip was applied to the lateral vaginal wall until it became wet,taking care to avoid cervical mucus, blood or semen that are known toaffect vaginal pH. The color of the strip was compared immediately witha colorimetric scale and the measurement was recorded.

During the gynecological examinations, the investigator performed avisual evaluation of vaginal mucosa using a four-point scale (0=none,1=mild, 2=moderate, and 3=severe) to assess parameters of vaginalsecretions, vaginal epithelial integrity, vaginal epithelial surfacethickness, and vaginal color according to the table below.

Assessment Severity Criteria No atrophy Mild Moderate Severe Vaginalnormal clear superficial coating of scant not covering none, inflamed,secretions secretions noted on secretions, difficulty the entire vaginalulceration noted, vaginal walls with speculum vault, may need needlubrication with insertion lubrication with speculum insertion tospeculum insertion prevent pain to prevent pain Vaginal normal vaginalsurface vaginal surface vaginal surface has epithelial bleeds withscraping bleeds with light petechiae before integrity contact contactand bleeds with light contact Vaginal rogation and poor rogation withsmooth, some smooth, no elasticity, epithelial elasticity of vault someelasticity noted elasticity of constriction of the surface of vaginalvault vaginal vault upper one third of thickness vagina or loss ofvaginal tone (cystocele and rectocele) Vaginal color pink lighter incolor pale in color transparent, either no color or inflamed

The VVA symptom self-assessment questionnaire, shown below, is aninstrument for subjects to self-assess their symptoms of vulvar orvaginal atrophy, including vaginal pain associated with sexual activity,vaginal dryness, vulvar or vaginal itching, or irritation. At screeningvisit 1A subjects were asked to complete the questionnaire and identifytheir most bothersome symptoms, and the results of the survey were usedto determine initial eligibility for the study. At visit 1A, subjectswere also asked to indicate which moderate or severe symptoms botheredthem most. The questionnaire was administered again at screening visit1B and used to determine continued eligibility for the study.

VVA SYMPTOMS SELF-ASSESSMENT Severity Score Please Rate your (Pleaseselect only ONE) Vulvar and/or Vaginal 3 = Symptoms 0 = None 1 = Mild 2= Moderate Severe 1 Pain associated with sexual activity (with vaginalpenetration). 2 Vaginal dryness. 3 Vulvar and/or vaginal itching orirritation.

Randomized subjects were asked to complete the VVA SymptomSelf-Assessment Questionnaire at visits 3, 4, 5, and 6. Subjects wereasked to indicate if no sexual activity with vaginal penetration wasexperience since the previous visit. Screening visit 1B evaluationresults were considered as Baseline data for the statistical analyses.

The Female Sexual Function Index (FSFI) is a brief, multidimensionalscale for assessing sexual function in women (see, Rosen, 2000, supra26: p. 191-208, incorporated by reference herein). The scale consists of19 items that assess sexual function over the past 4 weeks and yielddomain scores in six areas: sexual desire, arousal, lubrication, orgasm,satisfaction, and pain. Further validation of the instrument wasconducted to extend the validation to include dyspareunia/vaginismus(pain), and multiple sexual dysfunctions (see, Weigel, M., et al. “TheFemale Sexual Function Index (FSFI): Cross-Validation and Development ofClinical Cutoff Scores.” Journal of Sex & Marital Therapy, 2005. 31: p.1-20, incorporated by reference herein). The FSFI was conducted atVisits 2 and 6. Subjects participating in the PK substudy were notassessed using FSFI.

Safety Assessments

A complete medical history, including demographic data (age andrace/ethnicity) gynecological, surgical, and psychiatric history and useof tobacco and alcohol was recorded at the washout/screening visit 1Aprior to any washout period; this history included a review of all pastand current diseases and their respective durations as well as anyhistory of amenorrhea.

A complete physical examination was conducted at screening visit 1A andvisit 6/end of treatment. The physical examination included, at aminimum, examination of the subject's general appearance, HEENT (head,eyes, ears, nose, and throat), heart, lungs, musculoskeletal system,gastrointestinal (GI) system, neurological system, lymph nodes, abdomen,and extremities. The subject's height was measured at washout/screeningvisit 1A only and body weight (while the subject is lightly clothed) wasbe measured at washout/screening visit 1A and end of treatment. BMI wascalculated at washout/screening visit 1A. Vital signs (body temperature,heart rate [HR], respiration rate [RR], and sitting blood pressure [BP])were measured at all visits after the subject had been sitting for ≥10minutes. If the initial BP reading was above 140 mmHg systolic or 90mmHg diastolic, the option for a single repeat assessment performed 15minutes later was provided. A standard 12-lead ECG was obtained atscreening visit 1A and visit 6 or early termination.

Subjects were required to have a pelvic examination and Pap smearperformed during the screening visit 1B and visit 6 or earlytermination. The Pap smear was required for all subjects with or withoutan intact uterus and cervix. For subjects without an intact cervix thePap smear was obtained by sampling the apex of the vaginal cuff. Allsubjects were required to have a Pap smear done during screening,regardless of any recent prior assessment. Subjects who discontinued thestudy after 2 weeks of investigational product were required to have anend of treatment Pap smear. Subjects had a breast examination performedduring screening visit 1A and at visit 6 or early termination.

Endometrial biopsies were performed by a board-certified gynecologist atscreening and at visit 6/end of treatment. Unscheduled endometrialbiopsies were performed during the study, when indicated for medicalreasons. The screening biopsy was performed at screening visit 1B, afterthe subject's initial screening visit assessments indicated that thesubject was otherwise an eligible candidate for the study.

At screening, endometrial biopsies were read centrally by twopathologists. A candidate subject was excluded from the study if atleast one pathologist assessed the endometrial biopsy as endometrialhyperplasia, endometrial cancer, proliferative endometrium, weaklyproliferative endometrium, or disordered proliferative pattern, or if atleast one pathologist identified an endometrial polyp with hyperplasia,glandular atypia of any degree (e.g., atypical nuclei), or cancer.Additionally, identification of sufficient tissue to evaluate the biopsyby at least one pathologist was required for study eligibility. Theoption for one repetition of the screening endometrial biopsy was madeavailable when an initial endometrial biopsy was performed and both ofthe primary pathologists reported endometrial tissue insufficient fordiagnosis, no endometrium was identified, or no tissue was identified,and if the subject had met all other protocol-specified eligibilitycriteria to date. The visit 6 (or early termination) endometrialbiopsies and on treatment unscheduled biopsies were assessed by threepathologists.

During the study, at early termination, and at the end of the study, anysubject with a diagnosis of endometrial hyperplasia was withdrawn andtreated with 10 mg of Medroxyprogesterone acetate (MPA) for 6 monthsunless deemed otherwise by the PI. For unscheduled biopsies, thehistological diagnosis of endometrial polyp did not force withdrawalunless atypical nuclei were present.

A urine pregnancy test was conducted at screening visit 1A unless thesubject had a history of tubal ligation, bilateral oophorectomy, or was≥55 years of age and had experienced cessation of menses for at least 1year.

Blood samples for blood chemistry, hematology, coagulation tests, andhormone levels and urine samples for urine analysis were collected andsent to a central laboratory. Blood Chemistry (sodium, potassium,chloride, total cholesterol, blood urea nitrogen (BUN), iron, albumin,total protein, aspartate aminotransferase (AST), alanineaminotransferase (ALT), alkaline phosphatase, creatinine, calcium,phosphorous, uric acid, total bilirubin, glucose and triglycerides (mustbe fasting minimum of 8 hours). A fasting glucose of >125 mg/dL willrequire a HgA1C) was monitored. Hematology (complete blood count (CBC)including white blood cell count and differential, red blood cell count,hemoglobin, hematocrit, and platelet count) was monitored. HormoneLevels (follicle-stimulating hormone (FSH) (not required for subjectswith ≥12 months of spontaneous amenorrhea or bilateral oophorectomy),estradiol, estrone, and estrone conjugates and SHBG for subjects in thePK substudy) were monitored. Urine Analysis (appearance, specificgravity, protein, and pH) was conducted.

Pharmacokinetic Assessment

Seventy-two subjects were also enrolled in a pharmacokinetic (PK)substudy. In those subjects participating in the PK substudy, time 0 hserum blood samples were obtained at screening visit 1A, day 1, and day14 prior to dosing for baseline. The baseline was characterized by theaverage of the two pre-treatment samples. Serum blood samples were thenobtained on day 1 and day 14 at five post dose time points (2 h, 4 h, 6h, 10 h, and 24 h). On study days 1 (visit 2) and 14 (visit 3) abaseline pretreatment blood sample (Time 0 h) was collected from eachsubject prior to insertion of the investigational product. Afterinsertion of the product, blood samples were drawn at 2, 4, 6, 10, and24 hours following insertion. The last PK sample (approximately day 84)was obtained 4 days following the last insertion of investigationalproduct.

Blood samples were analyzed to characterize area under the curve (AUC),time of maximum concentration (t_(max)), minimum concentration(C_(max)), and maximum concentration (C_(max)). Blood samples were alsoanalyzed to measure the levels of estradiol, estrone, and estroneconjugates. No fasting requirements were applied. Sex hormone bindingglobulin (SHBG) levels were obtained at pre-treatment baseline (day 1,visit 2), and day 14 at the 0 h and on the day 84 final hormone blooddraw.

A symptoms/complaints and medications diary was dispensed at all visitsand subjects were instructed on completion. The subjects used the diaryto record symptoms/complaints (including stop and start dates andtreatment received) and prior medications/treatments (includingindication, stop, and start dates). A copy of the diary was made at eachvisit and re-dispensed to the subject. A dosing diary was dispensed atvisit 2 and at visit 3 and subjects were be instructed on completion.Subjects recorded investigational product usage and sexual activity. Thedosing diary dispensed at visit 3 was re-dispensed at visits 4 and 5. Acopy of the diary was made at each visit prior to re-dispensing to thesubject.

Study Visits

Study visits were typically conducted so as to include the activitiesoutlined in Table 43.

TABLE 43 Schedule of Assessments - Main Study Visit 2: Visit 3:Randomization/ Interim Washout Visit 1A Visit 1B Baseline Week 2 Week−14 to Screening Screening Week 0 Day 14 Activity −6 Week −6 to 0 Week−4 to 0 Day 1 (±3 d) Informed consent X X Demographics/Medical and X XGynecological history and prior medications Weight X X Height and BMIcalculation X X Vital signs X X X X X MBS X Subject VVA Self-AssessmentX X X Questionnaire Physical examination including breast X examLaboratory safety tests (Hematology, X Serum Chemistry, FSHP,Urinalysis) 12-Lead ECG X Pelvic exam X Vaginal pH X X Papanicolaou(Pap) smear X Investigator assessment of vaginal X X mucosa Vaginalcytological smear X X Mammogram X Endometrial biopsy X Diary Dispense XX X X X Diary Collection X X X X FSFI X Satisfaction Survey Urinepregnancy test X Randomization X Dispense Investigational Product bottleX Re-dispense Investigational Product X bottle Treatment administrationinstruction X X Collect unused investigational product X and usedbottles; assess compliance Adverse event monitoring X X X X Concomitantmedications X X X X Visit 6: End Telephone Visit 4: Visit 5: ofTreatment Interview Interim Interim or Early Week 14 Week 6 Week 8 Termapproximately Day 42 Day 56 Week 12 15 days after Activity (±3 d) (±3 d)Day 84 (±3 d) last dose of IP Informed consent Demographics/Medical andGynecological history and prior medications Weight X Height and BMIcalculation Vital signs X X X MBS Subject VVA Self- X X X AssessmentQuestionnaire Physical examination X including breast exam Laboratorysafety tests X (Hematology, Serum Chemistry, FSHP, Urinalysis) 12-LeadECG X Pelvic exam X Vaginal pH X X X Papanicolaou (Pap) smear XInvestigator assessment of X X X vaginal mucosa Vaginal cytologicalsmear X X X Mammogram Endometrial biopsy X Diary Dispense X X DiaryCollection X X X FSFI X Satisfaction Survey X Urine pregnancy testRandomization Dispense Investigational X Product bottle Re-dispenseInvestigational X Product bottle Treatment administration X Xinstruction Collect unused investigational X X X product and usedbottles; assess compliance Adverse event monitoring X X X X Concomitantmedications X X X X

Washout Period Visit (if applicable; Weeks −14 to −6). The purpose ofthis visit was to discuss the study with a potential subject and obtaininformed consent that is signed and dated before any procedures,including washout are performed. Subjects who agreed to discontinuecurrent treatment began washout after the consent form was signed. Asymptoms/complaints and medication diary was dispensed at this visit andthe subject was instructed in how to complete the diary. Once thewashout period was completed, the subject will return to the site forvisit 1A.

The activities and assessments conducted during the visit included:informed consent; demographics; medical/gynecological history;collection of prior and concomitant medication information; height, bodyweight measurement and BMI calculation; collection of vital signs (bodytemperature, HR, RR, and BP); dispensation of symptoms/complaints diaryand instruction in how to complete the diary

Screening Period Visits (Visits 1A and 1B). Subjects not requiringwashout begin screening procedures at visit 1A as described above forthe washout period. With the exception of vital signs, proceduresperformed at washout will not be repeated at screening visit 1A. Ingeneral, screening visits 1A and 1B were completed within 6 weeks (42days) for subjects without a uterus or within 8 weeks (56 days) forsubjects with a uterus. All screening assessments were completed priorto randomization. The investigators reviewed the results from allscreening procedures and determined if the subjects were eligible forenrollment into the study.

Visit 1A (approximately Week −6 to 0). Visit 1A was conducted after thewash-out period (if applicable) or after the subject provided informedconsent. The subject was advised to fast for 8 hours prior to the visitfor blood draws.

Procedures and evaluations conducted at the visit included: informedconsent; demographics; medical/gynecological history; collection of thesymptoms/complaints and medications diary from washout (if applicable)and review with the subject; recording of prior medication information;recording and assessment of adverse events (AEs) starting from thesigning of informed consent; height, body weight measurement and BMIcalculation; collection of vital signs (body temperature, HR, RR, andBP); physical examination; breast examination (including a mammogramconducted up to nine months prior to Visit 2); urine pregnancy test asrequired; blood and urine sample collection for blood chemistry (minimumfast of 8 hrs), hematology, and urinalysis; serum FSH as required;12-Lead ECG.

At visit 1A, the VVA symptom self-assessment questionnaire was conductedand most bothersome symptoms were identified, with the subjectself-identifying moderate or severe pain with sexual activity as her MBSto continue screening. The symptoms/complaints and medications diary wasdispensed, and subjects were instructed in how to complete the diary.Subjects were instructed to refrain from use of vaginal lubricants for 7days and sexual intercourse/vaginal douching for 24 hours prior to thevaginal pH assessment to be done at visit 1B.

Visit 1B (approximately Week −4 to Week 0). Visit 1B was conducted afterthe subject's initial screening visit and after the other screeningresults indicated that the subject was otherwise an eligible candidatefor the study (preferably around the middle of the screening period).

Procedures and evaluations conducted at the visit included: VVA symptomself-assessment questionnaire, the subject having indicated moderate tosevere pain with sexual activity with vaginal penetration in order tocontinue screening; collection of vital signs (body temperature, HR, RR,and BP); pelvic examination; investigator assessment of vaginal mucosaas described above; assessment of vaginal pH (sexual intercourse orvaginal douching within 24 hrs prior to the assessment being prohibited,and a subject's vaginal pH being >5.0 to continue screening); Pap smear;vaginal cytological smear (one repetition being permitted duringscreening if no results were obtained from the first smear); endometrialbiopsy performed as described above; review of the symptoms/complaintsand medications diary with the subject.

Visit 2 (Week 0; Randomization/Baseline). Subjects who met entrycriteria were randomized to investigational product at this visit.Procedures and evaluations conducted at the visit included:self-administration of FSFI by subjects not participating in the PKsubstudy; review of the symptoms/complaints and medications diary withthe subject; review of evaluations performed at screening visits andverification of present of all inclusion criteria and the absence of allexclusion criteria; collection of vital signs (body temperature, HR, RR,and BP); randomization, with subjects meeting all entry criteria beingrandomized and allocated a bottle number; dispensation ofinvestigational product and instruction in how to insert the capsulevaginally, with subjects receiving their first dose of investigationalproduct under supervision; dispensation of dosing diary and instructionon completion of the treatment diary, including recordinginvestigational product usage and sexual activity.

Visit 3 (Week 2, Day 14±3 days). Procedures and evaluations conducted atthe visit included: completion of the VVA symptom self-assessmentquestionnaire; review of the symptoms/complaints and medications diarieswith the subject; collection of vital signs (body temperature, HR, RR,and BP); Assessment of vaginal mucosa; assessment of vaginal pH (withsexual intercourse or vaginal douching within 24 hrs prior to theassessment being prohibited); vaginal cytological smear; collection ofunused investigational product and bottle for assessment ofcompliance/accountability; re-dispensation of investigational productand re-instruction in how to insert the capsule vaginally if necessary;review of the completed dosing diary with the subject.

Visit 4 (Week 6, Day 42±3 days). Procedures and evaluations conducted atthe visit included: completion of the VVA symptom self-assessmentquestionnaire; review of the symptoms/complaints and medications diarywith the subject; collection of vital signs (body temperature, HR, RR,and BP); assessment of vaginal mucosa as described above; vaginalcytological smear; assessment of vaginal pH (with sexual intercourse orvaginal douching within 24 hrs prior to the assessment beingprohibited); collection of unused investigational product for assessmentof compliance/accountability; re-dispensation of investigational productand re-instruction in how to insert the capsule vaginally if necessary;review of the completed dosing diary with the subject.

Visit 5 (Week 8, Day 56±3 days). Procedures and evaluations conducted atthe visit included: completion of the VVA symptom self-assessmentquestionnaire; review of the symptoms/complaints and medications diarywith the subject; collection of vital signs (body temperature, HR, RR,and BP); assessment of vaginal mucosa as described above; vaginalcytological smear; assessment of vaginal pH (with sexual intercourse orvaginal douching within 24 hrs prior to the assessment beingprohibited); collection of unused investigational product for assessmentof compliance/accountability; re-dispensation of investigational productand re-instruction in how to insert the capsule vaginally if necessary;review of the completed dosing diary with the subject.

Visit 6 (Week 12, Day 84±3 days or early termination). This visit wasperformed if a subject withdraws from the study before visit 6.Procedures performed at this visit included: completion of the VVAsymptom self-assessment questionnaire; review of the subject the dosingdiary, symptoms/complaints, and medications diaries with the subject;collection of blood and urine sample collection for blood chemistry(minimum fast of 8 hrs), hematology, and urinalysis; collection of vitalsigns (body temperature, HR, RR, and BP) and weight; performance of12-lead-ECG; collection of unused investigational product and containerfor assessment of compliance/accountability; physical examination;breast exam; assessment of vaginal mucosa as described above; assessmentof vaginal pH (with sexual intercourse or vaginal douching within 24 hrsprior to the assessment being prohibited); vaginal cytological smear;Pap smear; endometrial biopsy; self-administration of FSFI by subjectsnot participating in the PK substudy; self-administration of surveytitled “Acceptability of product administration Survey” by subjects.

Follow-up Interview (approximately 15 days after the last dose ofinvestigational product). Each subject who received investigationalproduct received a follow-up phone call, regardless of the duration oftherapy, approximately 15 days following the last dose ofinvestigational product. The follow-up generally took place afterreceipt of all safety assessments (e.g., endometrial biopsy andmammography results). The follow-up included: review of ongoing adverseevents and any new adverse events that occurred during the 15 daysfollowing the last dose of investigational product; review of ongoingconcomitant medications and any new concomitant medications thatoccurred during the 15 days following the last dose of investigationalproduct; and discussion of all end of study safety assessments anddetermination if further follow up or clinic visit is required.

PK Substudy Visit Procedures and Schedule

Screening Visit 1A. In addition to the procedures listed describedabove, activities in the PK substudy also included: provision ofinformed consent by subject and agreement to participate in the PKsubstudy; collection of a serum blood sample during the visit forbaseline assessment of estradiol, estrone, and estrone conjugates.

Visit 2 (Week 0, Day 1). In addition to the procedures listed describedabove, activities in the PK substudy also included collection of serumblood sample obtained prior to the administration of investigationalproduct (timepoint 0 h) for baseline assessment of estradiol, estrone,estrone conjugates, and SHBG. The investigational product wasself-administered by the subject after the pre-treatment blood samplehas been taken. After investigational product administration, serumblood samples were obtained at 2 h, 4 h, 6 h, 10 h, and 24 h timepointsfor estradiol, estrone, and estrone conjugates (serum samples weregenerally taken within +/−5 minutes at 2 h and 4 h, within +/−15 minutesat 6 h, and within +/−1 h at 10 h and 24 h). The subject was releasedfrom the site after the 10 hour sample and instructed to return to thesite the next morning for the 24 hour blood draw. The subject wasinstructed not to self-administer the day 2 dose until instructed by thesite personnel to dose at the clinical site. The subject was releasedfrom the clinical site following the 24 hour blood sample andadministration of the day 2 dose.

Visit 3 (Week 2, Day 14). The visit must occurred on day 14 with novisit window allowed. In addition to the procedures listed above, the PKsubstudy included collection of a serum blood sample prior to theadministration of day 14 dose (timepoint 0 h) for SHBG and PKassessments. The subject self-administered the day 14 dose at theclinical site, and serum blood samples were obtained at 2 h, 4 h, 6 h,10 h, and 24 h timepoints for estradiol, estrone, and estroneconjugates. The subject was released from the site after the 10 hoursample and instructed to return to the site the next morning for the 24hour blood draw. The subject was instructed not to self-administer theday 15 dose until instructed by the site personnel to dose at theclinical site. The subject was released from the clinical site followingthe 24 hour blood sample and administration of the day 15 dose. Thesubject was be instructed to administer the next dose of study drug onday 18 or day 19 and continue dosing on a bi-weekly basis at the sametime of day for each dose.

Visit 6 (Week 12, Day 84±3 days, or at early termination). The visittook place 4 days after last IP dose or early termination. A serumsample for estradiol, estrone, and estrone conjugates and SHBG was drawnin addition to the procedures described above.

PK sub-study visits were typically conducted so as to include theactivities outlined in Table 44.

TABLE 44 Schedule of Assessments for PK Sub-study Visit 2:Randomization/ Visit 3: Interim Washout Visit 1A Visit 1B Baseline Week2 Week −14 Screening Screening Week 0 Day 14 Activity to −6 Week −6 to 0Week −4 to 0 Day 1 (no window) PK sub-study Informed X X consentDemographics/Medical X X and Gynecological history and prior medicationsWeight X X Height and BMI X X calculation Vital signs X X X X X MBS XSubject VVA Self- X X X Assessment Questionnaire Physical examination Xincluding breast exam Laboratory safety tests X (Hematology, SerumChemistry, FSHP, Urinalysis) PK Serum Blood Samples X X X (Estradiol,Estrone, Estrone Conjugates) Serum blood samples for X X SHBG 12-LeadECG X Pelvic exam X Vaginal pH X X Papanicolaou (Pap) smear XInvestigator assessment of X X vaginal mucosa Vaginal cytological smearX X Mammogram X Endometrial biopsy X Diary Dispense X X X X X DiaryCollection X X X X Satisfaction Survey Urine pregnancy test XRandomization X Dispense new X Investigational Product (IP) bottleRe-dispense X Investigational Product (IP) bottle IP administration X Xinstruction Collect unused IP and X used bottles; assess complianceAdverse event monitoring X X X X Concomitant medications X X X X Visit6: End of treatment or Early Telephone Visit 4: Visit 5: Term InterviewInterim Interim Week 12 Week 14 Week 6 Week 8 Day 84 (±3 d)approximately Day 42 Day 56 (4 days after 15 days after Activity (±3 d)(±3 d) last IP dose) last dose of IP PK sub-study Informed consentDemographics/Medical and Gynecological history and prior medicationsWeight X Height and BMI calculation Vital signs X X X MBS Subject VVASelf- X X X Assessment Questionnaire Physical examination X includingbreast exam Laboratory safety tests X (Hematology, Serum Chemistry,FSHP, Urinalysis) PK Serum Blood X Samples (Estradiol, Estrone, EstroneConjugates) Serum blood samples X for SHBG 12-Lead ECG X Pelvic exam XVaginal pH X X X Papanicolaou (Pap) X smear Investigator assessment X XX of vaginal mucosa Vaginal cytological X X X smear MammogramEndometrial biopsy X Diary Dispense X X Diary Collection X X XSatisfaction Survey X Urine pregnancy test Randomization Dispense new XInvestigational Product (IP) bottle Re-dispense X InvestigationalProduct (IP) bottle IP administration X X instruction Collect unused IPand X X X used bottles; assess compliance Adverse event X X X Xmonitoring Concomitant X X X X medications

An Adverse Event (AE) in the study was defined as the development of anundesirable medical condition or the deterioration of a pre-existingmedical condition following or during exposure to a pharmaceuticalproduct, whether or not considered casually related to the product. AnAE could occur from overdose of investigational product. In this study,an AE can include an undesirable medical condition occurring at anytime, including baseline or washout periods, even if no study treatmenthas been administered. Relationship to Investigational Product

The investigators determined the relationship to the investigationalproduct for each AE (Not Related, Possibly Related, or ProbablyRelated). The degree of “relatedness” of the adverse event to theinvestigational product was described as follows: not related—notemporal association and other etiologies are likely the cause;possible—temporal association, but other etiologies are likely thecause. However, involvement of the investigational product cannot beexcluded; probable—temporal association, other etiologies are possiblebut unlikely. The event may respond if the investigational product isdiscontinued.

Example 11 Efficacy Results of Randomized, Double-Blind,Placebo-Controlled Multicenter study

Each of the three doses showed statistical significance compared withplacebo for the primary endpoints. Each of the three doses showedstatistical significance compared with placebo for the secondaryendpoints. Table 45 shows the statistical significance of theexperimental data for each of the four co-primary endpoints. Each of thedosages met each of the four co-primary endpoints at a statisticallysignificant level. The 25 mcg dose of TX-004HR demonstrated highlystatistically significant results at the p≤0.0001 level compared toplacebo across all four co-primary endpoints. The 10 mcg dose ofTX-004HR demonstrated highly statistically significant results at thep≤0.0001 level compared to placebo across all four co-primary endpoints.The 4 mcg dose of TX-004HR also demonstrated highly statisticallysignificant results at the p≤0.0001 level compared to placebo for theendpoints of superficial vaginal cells, parabasal vaginal cells, andvaginal pH; the change from baseline compared to placebo in the severityof dyspareunia was at the p=0.0255 level.

TABLE 45 Statistical Significance of Results for Co-Primary Endpoints(Based on Mean Change from Baseline to Week 12 Compared to Placebo) 25mcg 10 mcg 4 mcg Superficial Cells P < 0.0001 P < 0.0001 P < 0.0001Parabasal Cells P < 0.0001 P < 0.0001 P < 0.0001 Vaginal pH P < 0.0001 P< 0.0001 P < 0.0001 Severity of P = 0.0001 P = 0.0001 P = 0.0255Dyspareunia

Statistical improvement over placebo was also observed for all threedoses at the first assessment at week two and sustained through week 12.The pharmacokinetic data for all three doses demonstrated low systemicabsorption, supporting the previous Phase 1 trial data. TX-004HR waswell tolerated, and there were no clinically significant differencescompared to placebo-treated women with respect to adverse events. Therewere no drug-related serious adverse events reported.

As shown in the data below, in the MITT population (n=747) at week 12,all TX-004HR doses compared with placebo significantly decreased thepercentage of parabasal cells and vaginal pH, significantly increasedthe percentage of superficial cells, and significantly reduced theseverity of dyspareunia (all p≤0.00001 except dyspareunia at 4 μgp=0.0149).

At weeks 2, 6, and 8, the percentage of parabasal cells and vaginal pHsignificantly decreased p<0.00001); the percentage of superficial cellssignificantly increased (p<0.00001); and the severity of dyspareuniasignificantly improved from baseline with all TX-004HR doses vs placebo(4 μg p<0.03; 10 μg and 25 μg p<0.02).

Moderate-to-severe vaginal dryness was reported by 93% at baseline andsignificantly improved (p<0.02) for all doses at weeks 2, 6, 8, and 12(except 4 μg at week 2). Vulvar and/or vaginal itching or irritationsignificantly improved (p<0.05) for 10 μg at weeks 8 and 12, and for 25μg at week 12.

TX-004HR was well tolerated, had high acceptability, and notreatment-related serious AEs were reported in the safety population(n=764). There were no clinically significant differences in any AEs ortreatment-related SAEs between TX-004HR and placebo. Very low tonegligible systemic levels of estradiol were observed.

All TX-004HR doses were safe and effective and resulted in very low tonegligible systemic absorption of E2 in women with VVA andmoderate-to-severe dyspareunia. Onset of effect was seen as early as 2weeks and was maintained throughout the study and acceptability was veryhigh. This novel product provides a promising new treatment option forwomen experiencing menopausal VVA.

Cytology

Vaginal cytology data was collected as vaginal smears from the lateralvaginal walls according to procedures presented above to evaluatevaginal cytology at screening and Visit 6—End of treatment (day 84). Thechange in the Maturation Index was assessed as a change in cellcomposition measured at Visit 1—Baseline (day 1) compared to the cellcomposition measured at Visit 3—End of treatment (day 84). The change inpercentage of superficial, parabasal, and intermediate cells obtainedfrom the vaginal mucosal epithelium from a vaginal smear was recorded.Results from these assessments for superficial cells are presented inTable 46 and Table 47, as well as FIG. 10, FIG. 11, and FIG. 12. Resultsfrom these assessments for parabasal cells are presented in Table 48 andTable 49, as well as FIG. 13, FIG. 14, and FIG. 15.

Superficial Cells

TABLE 46 Superficial Cells P-values by Treatment Week 4 μg 10 μg 25 μgWeek 2 <0.0001 <0.0001 <0.0001 Week 6 <0.0001 <0.0001 <0.0001 Week 8<0.0001 <0.0001 <0.0001 Week 12 <0.0001 <0.0001 <0.0001

TABLE 47 Superficial Cells Change in Severity from Baseline by TreatmentWeek (change in percent of total vaginal cells) 4 μg 10 μg 25 μg PlaceboWeek 2 31.35(1.496) 31.93(1.488) 38.85(1.5) 6.05(1.498) Week 618.41(1.536) 16.88(1.543) 22.65(1.532) 5.43(1.525) Week 8 19.04(1.561)17.41(1.558) 23.88(1.554) 5.98(1.551) Week 12 17.5(1.542) 16.72(1.54)23.2(1.529) 5.63(1.537)

The study showed the formulations disclosed herein across all dosesincreased the percentage of superficial cells across all dosages in astatistically significant way.

Parabasal Cells

TABLE 48 Parabasal Cells P-values by Treatment Week 4 μg 10 μg 25 μgWeek 2 <0.0001 <0.0001 <0.0001 Week 6 <0.0001 <0.0001 <0.0001 Week 8<0.0001 <0.0001 <0.0001 Week 12 <0.0001 <0.0001 <0.0001

TABLE 49 Parabasal Cells Change in Severity from Baseline by TreatmentWeek (change in percent of total vaginal cells) 4 μg 10 μg 25 μg PlaceboWeek 2 −40.23(1.719) −44.42(1.708)  −45.6(1.723)   −7(1.72) Week 6−39.36(1.75)  −43.55(1.752) −45.61(1.746)  −9.23(1.741) Week 8−41.87(1.768) −43.78(1.764) −45.08(1.762) −7.86(1.76) Week 12−40.63(1.755) −44.07(1.751) −45.55(1.745) −6.73(1.75)

The increase of superficial cells and decrease of parabasal cells showedstatistical significance over placebo at week 2 and for every weekthereafter, including at week 12. Administration of the pharmaceuticalformulation resulted in rapid onset of action, as early as two weeksafter the initial administration. Rapid onset of action may be coupledwith the rapid absorption demonstrated in the pharmacokinetic datapresented below.

pH

Vaginal pH was measured at Screening and Visit 6—End of treatment (day84). The pH measurement was obtained as disclosed herein. Results fromthese assessments are presented in Table 50 and Table 51, and FIG. 16,FIG. 17, and FIG. 18.

TABLE 50 pH P-values by Treatment Week 4 μg 10 μg 25 μg Week 2 <0.0001<0.0001 <0.0001 Week 6 <0.0001 <0.0001 <0.0001 Week 8 <0.0001 <0.0001<0.0001 Week 12 <0.0001 <0.0001 <0.0001

TABLE 51 pH Change in Severity from Baseline by Treatment Week (changein pH) 4 μg 10 μg 25 μg Placebo Week 2 −1.23(0.064) −1.37(0.064) −1.3(0.065) −0.28(0.064) Week 6 −1.32(0.066)  −1.4(0.066) −1.48(0.066) −0.3(0.065) Week 8 −1.35(0.067) −1.46(0.067) −1.45(0.066) −0.38(0.066)Week 12 −1.32(0.066) −1.42(0.066) −1.34(0.066) −0.28(0.066)

The decrease in vaginal pH was observed at statistically significantlevels at week 2 and through the end of the study. Surprisingly, the pHdecreased in all three pharmaceutical formulations tested and at allthree dosages of over a full pH unit for all three doses.

Most Bothersome Symptoms

Dyspareunia

Subjects were asked to specify the symptom that she identified as the“most bothersome symptom.” During the screening period all of thesubjects were provided with a questionnaire to self-assess the symptomsof VVA: (1) dyspareunia; (2) vaginal dryness; and (3) vaginal or vulvarirritation, burning, or itching. Each symptom was measured on a scale of0 to 3, where 0=none, 1=mild, 2=moderate, and 3=severe. Each subject wasgiven a questionnaire at each visit and the responses were recorded. Allrandomized subjects were also provided a questionnaire to self-assessthe symptoms of VVA at Visit 1 and on each subsequent visit throughVisit 6—End of the treatment (day 84). Subjects recorded theirself-assessments daily in a diary and answers were collected on visits 8and 15 (end of treatment). Pre-dose evaluation results obtained at Visit1 were considered as baseline data for the statistical analyses. Datafrom these assessments for dyspareunia are presented in Table 52 andTable 53. Data from these assessments for dryness are presented in Table54 and Table 55.

TABLE 52 Dyspareunia P-values by Treatment Week 4 μg 10 μg 25 μg Week 20.026 0.0019 0.0105 Week 6 0.0069 0.0009 <0.0001 Week 8 0.0003 <0.0001<0.0001 Week 12 0.0149 <0.0001 <0.0001

TABLE 53 Dyspareunia Change in Severity from Baseline by Treatment Week(0 to 3 severity scale) 4 μg 10 μg 25 μg Placebo Week 2 −0.99(0.072)−1.08(0.072) −1.02(0.073) −0.76(0.072) Week 6  −1.3(0.072) −1.37(0.072)−1.48(0.072) −1.03(0.07)  Week 8 −1.52(0.073) −1.64(0.074) −1.62(0.075)−1.15(0.072) Week 12 −1.52(0.071) −1.69(0.071) −1.69(0.071) −1.28(0.07) 

Each of the 4 μg, 10 μg, and 25 μg formulations tests demonstrated anearly onset of action at week 2 for the most bothersome symptom ofdyspareunia, evidenced by the statistically significant results(measured by p-value) in Table 52. After two weeks, each dosedemonstrated separation from placebo in improvement in the mostbothersome symptom of dyspareunia.

Coupled with the PK data presented below, these results show that theformulations disclosed herein provide a bolus of estradiol within twohours of administration, which resulted in a decrease in the severity ofdyspareunia as early as two weeks later. Estradiol is rapidly absorbedat around two hours, which is significantly faster than the formulationsof the prior art that sought an extended release profile. The rapidabsorption of estradiol is believed to be a result of administrationwith a liquid formulation.

Surprisingly, the 4 μg formulation showed clinical effectiveness at twoweeks along with the 25 μg and 10 μg dosage levels. These datademonstrate that 4 μg is an effective dose, and can be effective asearly as two weeks after administration for the most bothersome symptomof dyspareunia.

Dryness

TABLE 54 Dryness P-values by Treatment Week 4 μg 10 μg 25 μg Week 20.1269 0.0019 0.0082 Week 6 0.0094 0.0001 0.0005 Week 8 0.0128 <0.00010.0008 Week 12 0.0014 <0.0001 <0.0001

TABLE 55 Dryness Change in Severity from Baseline by Treatment Week (0to 3 severity scale) 4 μg 10 μg 25 μg Placebo Week 2 −0.86(0.066)−1.01(0.065) −0.96(0.066) −0.72(0.066) Week 6 −1.14(0.067) −1.27(0.068)−1.23(0.067)  −0.9 (0.067) Week 8 −1.25(0.069) −1.44(0.068) −1.34(0.068)−1.01(0.068) Week 12 −1.27(0.068) −1.47(0.067) −1.47(0.067) −0.97(0.067)

Each of the 4 μg, 10 μg, and 25 μg formulations tests demonstrated anearly onset of action at week 2 for the most bothersome symptom ofdryness, evidenced by the statistically significant results (measured byp-value) in Table 54. After two weeks, each dose demonstrated separationfrom placebo in improvement in the most bothersome symptom of dryness.

Irritation/Itching

TABLE 56 Irritation/Itching P-values by Treatment Week 4 μg 10 μg 25 μgWeek 2 0.9616 0.2439 0.6518 Week 6 0.7829 0.2328 0.4118 Week 8 0.06390.0356 0.0914 Week 12 0.0503 0.0055 0.0263

TABLE 57 Irritation/Itching Change in Severity from Baseline byTreatment Week (0 to 3 severity scale) 4 μg 10 μg 25 μg Placebo Week 2−0.47(0.054) −0.56(0.053) −0.51(0.054) −0.47(0.054) Week 6 −0.57(0.055)−0.64(0.055) −0.61(0.055) −0.55(0.055) Week 8 −0.74(0.056) −0.76(0.056)−0.73(0.056) −0.59(0.056) Week 12 −0.75(0.055) −0.81(0.055) −0.77(0.055) −0.6(0.055)

Vulvar and/or vaginal itching or irritation significantly improved(p<0.05) for 10 μg at weeks 8 and 12, and for 25 μg at week 12.Moreover, the trend for 4 μg was an improvement in itching week overweek to nearly being statistically significant at week 12.

Coupled with the PK data presented below, these results show that theformulations disclosed herein provide a bolus of estradiol within twohours of administration, which resulted in a decrease in the severity ofdryness as early as two weeks later. Estradiol is rapidly absorbed ataround two hours, which is significantly faster than the formulations ofthe prior art that sought an extended release profile. The rapidabsorption of estradiol is believed to be a result of administrationwith a liquid formulation.

Surprisingly, the 4 μg formulation showed clinical effectiveness at twoweeks along with the 25 μg and 10 μg dosage levels. These datademonstrate that 4 μg is an effective dose, and can be effective asearly as two weeks after administration for the most bothersome symptomof dryness.

As described above, each dose was compared with placebo for change frombaseline to week 12 in the percentages of vaginal superficial cells andparabasal cells, vaginal pH, and severity of dyspareunia (co-primaryendpoints). The proportion of responders (defined as women with ≥2 ofthe following at week 12: vaginal superficial cells >5%, vaginal pH<5.0,≥1 category improvement from baseline dyspareunia score) was compared inTX-004HR groups vs placebo. Pre-specified subgroup analyses ofco-primary endpoints were analyzed by age (≤56 years, 57-61 years, and≥62 years), BMI (≤24 kg/m², 25-28 kg/m², and ≥29 kg/m²), uterine status,parity, and vaginal births. Pharmacokinetic (PK) parameters werecompared with placebo in a sub-analysis of the main study.

The proportion of responders was significantly higher for all TX-004HRdose groups vs placebo (p<0.0001 for all). All TX-004HR doses vs placebosignificantly improved percentage of superficial and parabasal cells,vaginal pH, and severity of dyspareunia at 12 weeks. Subgroup analysesshowed generally similar results for percentage of superficial andparabasal cells and vaginal pH irrespective of age, BMI, uterine status,parity, and vaginal births. Severity of dyspareunia was significantlyreduced at 12 weeks with all TX-004HR doses vs placebo in most subgroups(Table 57A).

The PK sub-analysis (n=72), described in more detail below, found AUCand C_(avg) parameters for E2 and estrone (E1) with 4 μg and 10 μgTX-004HR to be similar to placebo. Increases occurred in E2 AUC andC_(avg) with 25 μg vs placebo but remained within the normalpostmenopausal range. E2 levels at day 84 were similar between theTX-004HR groups and placebo, indicating no systemic drug accumulation.

All doses of TX-004HR were associated with robust efficacy anddemonstrated a statistically significant difference vs placebo forincreasing superficial cells, decreasing parabasal cells and vaginal pH,and reducing the severity of dyspareunia. Age, BMI, uterine status,parity and vaginal births generally did not affect TX-004HR efficacy.These results occurred with negligible systemic absorption of TX-004HRestradiol doses of 4 μg, 10 μg, and 25 μg.

TABLE 57A Change from baseline to week 12 in the severity of dyspareunia(LS mean change ± SE). TX-004HR TX-004HR TX-004HR Placebo 4 μg 10 μg 25μg Key clinical factors (n = 187) (n = 186) (n = 188) (n = 186) Age,years ≤56 n = 52 −1.25 ± 0.119 n = 50 −1.58 ± 0.122 n = 61 −1.77 ±0.112^(†) n = 65 −1.86 ± 0.108^(‡) 57-61 n = 53 −1.39 ± 0.118 n = 50−1.42 ± 0.121 n = 49 −1.63 ± 0.121 n = 47 −1.79 ± 0.125* ≥62 n = 58−1.19 ± 0.122 n = 51 −1.52 ± 0.126 n = 44 −1.66 ± 0.138^(†) n = 47 −1.38± 0.135 BMI, ≤24 n = 56 −1.14 ± 0.115 n = 58 −1.48 ± 0.113* n = 56 −1.6± 0.117^(†) n = 51 −1.72 ± 0.123^(‡) kg/m² 25 to 28 n = 57 −1.48 ± 0.118n = 45 −1.51 ± 0.131 n = 52 −1.78 ± 0.124 n = 58 −1.77 ± 0.117 ≥29 n =50 −1.21 ± 0.125 n = 48 −1.56 ± 0.125 n = 46 −1.71 ± 0.129^(†) n = 50−1.57 ± 0.124* Uterine Intact n = 101 −1.35 ± 0.086 n = 82 −1.66 ±0.095* n = 84 −1.74 ± 0.095^(†) n = 85 −1.81 ± 0.094^(‡) statusNon-intact n = 62 −1.15 ± 0.115 n = 69 −1.35 ± 0.108 n = 70 −1.63 ±0.108^(†) n = 74 −1.55 ± 0.107* Pregnancy Pregnancy = 0 n = 16 −1.18 ±0.220 n = 17 −1.28 ± 0.217 n = 19 −1.26 ± 0.209 n = 13 −1.64 ± 0.257status Pregnancy ≥1 n = 147 −1.28 ± 0.073 n = 134 −1.55 ± 0.075* n = 135−1.74 ± 0.076^(§) n = 146 −1.70 ± 0.073^(‡) Vaginal Vaginal birth = 0 n= 26 −1.19 ± 0.171 n = 22 −1.74 ± 0.189* n = 29 −1.68 ± 0.161* n = 31−1.76 ± 0.160* births Vaginal birth ≥1 n = 121 −1.30 ± 0.080 n = 112−1.51 ± 0.082 n = 106 −1.77 ± 0.085^(‡) n = 115 −1.69 ± 0.082^(‡) *p <0.05; ^(†)p < 0.01; ^(‡)p < 0.001; ^(§)p < 0.0001 vs placebo.

Visual evaluation of the vaginal epithelium, a secondary endpoint of thetrial, was performed during gynecological examinations at baseline andweeks 2, 6, 8, and 12. A four-point score (0=none, 1=mild, 2=moderate,3=severe) was used to assess changes in vaginal color, vaginalepithelial integrity, vaginal epithelial surface thickness, and vaginalsecretions. Change from baseline to each time point was compared withplacebo using the mixed effect model repeat measurement (MMRM) analysis.

At baseline, women had mean scores of 1.8 for vaginal color, 1.5 forepithelial integrity, 1.9 for epithelial surface thickness, and 1.7 forsecretions. These scores were consistent with VVA reflecting pallor,diminished vaginal wall integrity and thickness, and secretions.Significant improvements from baseline at weeks 2, 6, 8 and 12 (Table57B; FIG. 19A-FIG. 19D) were observed for all 3 doses of TX-004HRcompared with placebo in vaginal color (white to pink), epithelialintegrity, epithelial surface thickness and secretions (p<0.001 forall). After 12 weeks, women in the active TX-004HR treatment groups hadmean scores less than 1 in all four characterized categories. Vaginalvisual examination of women in the 3 TX-004HR groups had greaterreported improvements from baseline in all vaginal parameters examinedthan placebo subjects and at all time points. These improved vaginalvisual scores reflect other observed measures of efficacy of TX-004HR (4μg, 10 μg, and 25 μg) at treating moderate-to-severe VVA inpostmenopausal women, with negligible to very low systemic E2absorption.

TABLE 57B Change from baseline at week 12 in vaginal parameters VaginalParameters, mean (SD) TX-004HR TX-004HR TX-004HR 4 μg 10 μg 25 μgPlacebo (n = 171) (n = 173) (n = 175) (n = 175) Vaginal epithelialBaseline 1.8 (0.61) 1.7 (0.59) 1.8 (0.60) 1.7 (0.64) color 12 weeks 0.8(0.67) 0.7 (0.64) 0.8 (0.68) 1.2 (0.80) Change −1.0 (0.82)   −1.1(0.80)   −1.0 (0.88)   −0.6 (0.83)   LS Mean (SE) −0.97 (0.05)*   −1.06(0.05)*   −0.96 (0.05)*   −0.60 (0.05)    Vaginal epithelial Baseline1.6 (0.84) 1.4 (0.83) 1.5 (0.77) 1.5 (0.84) integrity 12 weeks 0.5(0.69) 0.4 (0.57) 0.5 (0.66) 0.9 (0.91) Change −1.0 (0.93)   −1.0(0.89)   −1.0 (0.91)   −0.6 (0.98)   LS Mean (SE) −0.97 (0.05)*   −1.07(0.05)*   −1.01 (0.05)*   −0.60 (0.05)    Vaginal epithelial Baseline1.9 (0.67) 1.8 (0.63) 1.9 (0.59) 1.9 (0.65) surface thickness 12 weeks0.9 (0.66) 0.8 (0.63) 0.9 (0.69) 1.3 (0.85) Change −1.0 (0.76)   −1.0(0.79)   −0.9 (0.80)   −0.6 (0.82)   LS Mean (SE) −0.98 (0.05)*   −1.03(0.05)*   −0.94 (0.05)*   −0.61 (0.05)    Vaginal Baseline 1.8 (0.68)1.7 (0.66) 1.7 (0.63) 1.8 (0.63) secretions 12 weeks 0.8 (0.69) 0.6(0.67) 0.7 (0.71) 1.1 (0.84) Change −1.0 (0.82)   −1.0 (0.86)   −1.0(0.85)   −0.7 (0.79)   LS Mean (SE) −1.01 (0.05)*   −1.06 (0.05)*  −1.04 (0.05)*   −0.64 (0.05)    Data is mean (SD) unless otherwisenoted; *MMRM p < 0.0001 vs placebo.

A direct correlation was observed between the total sum of theindividual visual examination score and severity of dyspareunia (r=0.31;P<0.0001) as well as the severity of vaginal dryness (r=0.38; P<0.0001)at 12 weeks when all subjects were analyzed independent of treatment.See, FIG. 20A and FIG. 20B. Interestingly, women treated with placeboalso showed some improvements in their scores at week 2, but while womentreated with TX-004HR showed continued improvements through 12 weeks oftreatment, such continued improvements were not observed to the sameextent with the placebo. Three possible explanations for theimprovements observed with the placebo include the potential lubricatingeffect of the excipient Miglyol, a fractionated coconut oil contained inall softgel capsules, improved appearance based on vaginal lubricationcaused by increased sexual activity and/or bias on the part of thephysicians performing the examinations as they may anticipateimprovement. Nevertheless, TX-004HR still significantly improvedevaluated signs and symptoms of VVA better than placebo.

Since visual inspection of the vagina with the 4-point assessment toolpositively correlated with dyspareunia and vaginal dryness in thisstudy, this tool may help healthcare professionals diagnose VVA andassess its treatment, and provide a vehicle for health careprofessionals to initiation discussion with their patients about asensitive topic. Several large-scale studies have shown that it isdifficult for patients to discuss vulvovaginal health openly with theirhealth care professionals because they are either embarrassed,uninformed about VVA and its treatments, or believe that the topic isnot appropriate for discussion. Therefore, of the 50% of postmenopausalwomen who have symptoms of VVA, far fewer seek treatment. Visualexamination of the vagina may help practitioners identify women at riskof dyspareunia and vaginal dryness, and allow them to proactively engagewomen in conversations about VVA symptoms such as dyspareunia anddryness and discuss available treatment options.

Example 12 Pharmacokinetics Results in Randomized, Double-Blind,Placebo-Controlled Multicenter Study

While some approved local estrogens effectively treat VVA, systemicestradiol may increase with local administration. TX-004HR is a newlow-dose vaginal softgel capsule containing solubilized naturalestradiol designed to provide excellent efficacy with negligiblesystemic absorption. Up to three times lower systemic estrogen levelswere previously reported with TX-004HR vs an approved low-dose vaginalestradiol tablet. The present studies show that VVA efficacy can beachieved with negligible systemic absorption as measured by PK inpostmenopausal women with moderate-to-severe dyspareunia.

The terms “minimal systemic effect,” “low systemic absorption,” and“negligible systemic absorption,” as used herein, mean that thedisclosed formulations and methods result in low to minimal absorptionof estradiol in women, especially women with VVA and/or dyspareunia. Infact, it has surprisingly been found that the disclosed formulations andmethods result in negligible to very low systemic absorption ofestradiol, which remains in the postmenopausal range. The finding isborne out by the examples provided herein that demonstrate that theC_(max) and AUC levels of estradiol relative to placebo were notstatistically differentiable, which indicates that the formulationsdisclosed herein have a negligible systemic effect. As such, thedisclosed formulations and methods advantageously provide local benefitsin patients with VVA and/or dyspareunia (i.e., the disclosedformulations are extremely effective in increasing the superficialcells, decreasing parabasal cells, and decreasing pH) without increasingsystemic levels.

A PK substudy was part of a large, multicenter, double-blind,randomized, placebo-controlled phase 3 trial evaluating the efficacy andsafety of TX-004HR (4 μg, 10 μg, and 25 μg) compared with placebo fortreating postmenopausal moderate-to-severe dyspareunia. Women receivedTX-004HR or placebo once daily for 2 weeks then twice weekly for 10 wks.

In this study, the systemic exposure to estradiol following once dailyintravaginal administration of estradiol 25 μg, 10 μg, 4 μg, and placebowere investigated on days 1, 14, and 84 as described herein. Descriptivestatistics of the plasma estradiol concentrations taken at each samplingtime and the observed C_(max) values were recorded, as shown in thetables below and FIG. 21 and FIG. 22, for estradiol, estrone, andestrone conjugates for all three doses. Serum estradiol, estrone,estrone conjugates, and sex hormone binding globulin were measured.

For PK, serum was sampled pre-dose and at 2, 4, 6, 10, and 24 hpost-dose on days 1 and 14 for estradiol, estrone (E1), and estroneconjugates (E1Cs). Baseline-adjusted results are shown here; unadjusteddata will be presented. Efficacy endpoints were change from baseline toweek 12 for vaginal superficial cells (%), vaginal parabasal cells (%),vaginal pH, and severity of dyspareunia. Secondary endpoints wereseverity of dryness and itching/irritation. Blood chemistry was testedat week 12.

The substudy randomized 72 women (mean age 59 y) at 11 centers. Meanarea under the concentration-time curve (AUC) and average concentration(C_(avg)) for estradiol were not significantly different vs placebo with4 μg and 10 μg TX-004HR, but were significantly higher with 25 μg at day1 (AUC 130 vs 13.8 h*pg/mL and Cavg 5.4 vs 0.4 pg/mL) and day 14 (AUC84.6 vs 7.1 h*pg/mL and Cavg 3.5 vs −0.2 pg/mL).

Mean estradiol peak concentration (C_(max)) was not significantlydifferent with 4 μg (day 1: 2.6 pg/mL; day 14: 1.3 pg/mL) vs placebo(day 1: 2.1 pg/mL; day 14: 1.0 pg/mL), and although significant, wasnegligible with 10 μg (day 1: 6.0 pg/mL; day 14: 3.0 pg/mL) and very lowfor 25 μg (day 1: 26.2 pg/mL; day 14: 12.0 pg/mL).

E1 and E1Cs AUC, C_(avg), C_(max), C_(min) did not differ vs placebo,except for E1Cs on day 1 when AUC was significantly higher with 25 μg(2454 vs 83.0 h*pg/mL), C_(max) with 10 μg and 25 μg (90.2 and 198.6pg/mL, respectively vs 27.1 pg/mL), and C_(avg) with 10 μg (8.0 vs −33.7pg/mL).

In the overall study TX004-HR showed robust efficacy for symptoms ofdyspareunia, vaginal dryness and irritation at 12 weeks with all 3 dosescompared with placebo.

Vaginal TX-004HR resulted in negligible to very low systemic absorptionof estradiol, which remained in the postmenopausal range. TX-004HRimproved the signs and symptoms of VVA. This study supports localbenefits of estradiol without increasing systemic exposure.

The pharmacokinetic data for estradiol demonstrates the rapid absorptionof the formulations disclosed herein for all three doses. Surprisingly,while the pharmacokinetic data was extremely low for all three doses,each dose was extremely effective in increasing the superficial cells,decreasing parabasal cells, and decreasing pH.

The pharmaceutical compositions disclosed herein provide an improvedsafety profile over other options for treating VVA. The combination oflow systemic estradiol, while retaining efficacy was a surprising resultfor all three doses.

Estradiol Concentration

TABLE 58 Pharmacokinetics Estradiol Baseline (pg/mL) 4 μg 10 μg 25 μgPlacebo Baseline 4.7(4.41) 5(3.52) 3.6(1.86) 4.6(2.56)

TABLE 59 Pharmacokinetics Estradiol Day 1 (pg/mL) 4 μg 10 μg 25 μgPlacebo Predose 3.1(1.56) 4.9(3.47) 3.6(1.81) 4.1(2.45) 2 hour 6.1(2.3) 10.4(4.89)  28.7(17.91) 4.8(3.33) 4 hour 4.3(1.68) 6.7(3.59) 16.1(14.75)  5(3.59) 6 hour 3.7(1.96) 5.7(3.16) 9.7(6.86) 4.8(3.53) 10 hour3.7(1.47) 5.5(2.92) 6.2(2.37) 5.2(3.61) 24 hour 4.2(2.02) 5.4(4.44)6.2(8.43) 5.1(4.42)

TABLE 60 Pharmacokinetics Estradiol Day 14 (pg/mL) 4 μg 10 μg 25 μgPlacebo Predose 3.5(1.63) 3.8(2.56) 5.2(2.89) 4.2(3.07) 2 hour 4.3(2.01)6.3(2.29) 15.3(7.72)  4.2(2.44) 4 hour  4(1.7) 5.9(2.55)  11(4.86)4.7(3.2)  6 hour 3.9(1.92) 5.1(2.32) 7.9(3.35) 4.7(2.97) 10 hour3.8(2.12) 5(3)  6.8(3.76) 5.1(3.53) 24 hour 3.6(1.89) 3.7(2.05)4.9(4.35) 3.9(2.43)

TABLE 61 Pharmacokinetics Estradiol End of Study (pg/mL) 4 μg 10 μg 25μg Placebo Post Dosing 4.3(2.69) 4.8(2.57) 6.7(11.51) 4.4(2.6)

Estradiol Area Under the Curve (0-24 Hours)

TABLE 62 Estradiol Area Under the Curve (0-24 hours) (h*pg/mL) 4 μg 10μg 25 μg Placebo Day 1 91.7(37.86) 138.2(75.22) 217.4(99.02)116.6(77.3)  Day 14 87.2(42.77) 110.1(54.57) 171.6(80.13) 104.2(66.39)

TABLE 63 Estradiol Area Under the Curve (0-24 hours) (Baseline Adjusted)(h * pg/mL) 4 μg 10 μg 25 μg Placebo Day 1  12 (13.89) 21.9 (19.16)130.4 (111.95) 13.8 (28.86) Day 14 7.2 (12.08) 13.7 (18.77) 84.6 (62.7)  7.1 (20.28)

TABLE 64 Estradiol Area Under the Curve (0-24 hours) P-values PairwiseTest vs. 4 μg 10 μg 25 μg Day 1 0.0242 <0.0001 Day 14 0.1777 0.0005

TABLE 65 Estradiol Area Under the Curve (0-24 hours) P-values PairwiseTest vs. Placebo 4 μg 10 μg 25 μg Day 1 0.2292 0.4028 0.0021 Day 140.3829 0.7724 0.0108

TABLE 66 Estradiol Area Under the Curve (0-24 hours) P-values PairwiseTest vs. 4 μg (Baseline Adjusted) 10 μg 25 μg Day 1 0.082 0.0001 Day 140.2373 <0.0001

TABLE 67 Estradiol Area Under the Curve (0-24 hours) P-values PairwiseTest vs. Placebo (Baseline Adjusted) 4 μg 10 μg 25 μg Day 1 0.81340.3238 0.0002 Day 14 0.979 0.3235 <0.0001

TABLE 68 Estradiol Area Under the Curve (0-24 hours) Ratio (Day 14) ofDay 14 to Day 1 4 μg 10 μg 25 μg Placebo AUC Ratio of Day 14 0.971(0.2358) 0.876 (0.1937) 0.955 (0.6633) 0.949 (0.225) to Day 1 Pairwisetest vs 4 μg — 0.2022 0.9246 — Pairwise test vs 0.7859 0.3101 0.9748 —Placebo

Estradiol C_(max)

TABLE 69 C_(max) (pg/mL) 4 μg 10 μg 25 μg Placebo Day 1 6.5 (2.13) 10.9(5)     29.8 (17.51) 6.6 (4.85) Day 14 4.8 (2.31) 7.3 (2.36) 15.7 (7.61)5.5 (3.43)

TABLE 70 C_(max) (Baseline Adjusted) (pg/mL) 4 μg 10 μg 25 μg PlaceboDay 1 2.6 (2.17) 6 (4.44) 26.2 (18.19) 2.1 (3.48) Day 14 1.3 (1.08) 3(1.73)  12 (7.32)   1 (1.81)

TABLE 71 C_(max) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.0013 <0.0001 Day 14 0.0033 <0.0001

TABLE 72 C_(max) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μg Day1 0.9586 0.0116 <0.0001 Day 14 0.5174 0.0702 <0.0001

TABLE 73 C_(max) P-values Pairwise Test vs. 4 μg (Baseline Adjusted) 10μg 25 μg Day 1 0.0055 <0.0001 Day 14 0.002 <0.0001

TABLE 74 C_(max) P-values Pairwise Test vs. Placebo (Baseline Adjusted)4 μg 10 μg 25 μg Day 1 0.6074 0.0059 <0.0001 Day 14 0.5088 0.0022<0.0001

TABLE 75 C_(max) Ratio (Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μgPlacebo C_(max) Ratio of Day 14 to 0.77 (0.2633) 0.804 (0.3245) 0.929(1.5011) 0.933 (0.2406) Day 1 Pairwise test vs vs 4 μg — 0.7399 0.6702 —Pairwise test vs 0.0702 0.1946 0.9931 — Placebo

Estradiol C_(avg)

TABLE 76 C_(avg) (pg/mL) 4 μg 10 μg 25 μg Placebo Day 1 3.9 (1.46) 5.8(3.13) 9.1 (4.13) 4.9 (3.22) Day 14 3.6 (1.78) 4.6 (2.27) 7.1 (3.34) 4.3(2.77)

TABLE 77 C_(avg) (Baseline Adjusted) (pg/mL) 4 μg 10 μg 25 μg PlaceboDay 1   0 (1.93) 0.8 (0.95) 5.4 (4.66)  0.4 (1.35) Day 14 0.1 (0.68) 0.2(1.22) 3.5 (2.61) −0.2 (1.28)

TABLE 78 C_(avg) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.0294 <0.0001 Day 14 0.1777 0.0005

TABLE 79 C_(avg) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μg Day1 0.267 0.4028 0.0021 Day 14 0.3829 0.7724 0.0108

TABLE 80 C_(avg) P-values Pairwise Test vs. 4 μg (Baseline Adjusted) 10μg 25 μg Day 1 0.1076 0.0001 Day 14 0.7759 <0.0001

TABLE 81 C_(avg) P-values Pairwise Test vs. Placebo (Baseline Adjusted)4 μg 10 μg 25 μg Day 1 0.5126 0.2564 0.0001 Day 14 0.4098 0.3629 <0.0001

TABLE 82 C_(avg) Ratio (Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μgPlacebo C_(avg) Ratio of 0.77(0.2633) 0.804(0.3245) 0.929(1.5011)0.933(0.2406) Day 14 to Day 1 Pairwise test vs — 0.7399 0.6702 — vs 4 μgPairwise test vs 0.0702 0.1946 0.9931 — Placebo

Estradiol T_(max)

TABLE 83 T_(max) (h) 4 μg 10 μg 25 μg Placebo Day 1  7(9.36) 6.1(8.04)4.6(7.09) 8.6(6.74) Day 14 9.3(8.86)  4(2.57) 2.7(1.94) 7.2(3)  

TABLE 84 T_(max) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.7566 0.3834 Day 14 0.0206 0.004

TABLE 85 T_(max) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μg Day1 0.5705 0.3255 0.0943 Day 14 0.3576 0.0019 <0.0001

Estrone Concentration

TABLE 86 Pharmacokinetics Estrone Baseline (pg/mL) 4 μg 10 μg 25 μgPlacebo Baseline 15.9(6.02) 19.7(9.18) 16.3(7.71) 20.4(9.67)

TABLE 87 Pharmacokinetics Estrone Day 1 (pg/mL) 4 μg 10 μg 25 μg PlaceboPredose 14.7(4.44)  21(8.51) 17.2(8.5)  18.3(8.54)   2 hour 13.3(4.52) 20(8.53) 18.9(6.7)  18.9(11.25)  4 hour  13(4.68) 19.3(7.4)  19.4(7.06)19.9(13.87)  6 hour 13.9(6.04) 19.6(8.89) 19.1(8.1)   19(11.69) 10 hour13.4(4.94) 19.7(8.53) 18.8(7.18) 19.3(11.65) 24 hour 14.3(5.92)21.2(9.89) 16.6(6.06) 22.9(17.18)

TABLE 88 Pharmacokinetics Estrone Day 14 (pg/mL) 4 μg 10 μg 25 μgPlacebo Predose 15.8(5.15)  21.7(14.25) 18.6(8.49) 18.7(9.38)  2 hour13.6(5.3)  19.7(10.2) 19.8(9.08) 17.3(7.99)  4 hour  14(5.25)  21(13.46) 19.9(7.26)  20.4(11.41)  6 hour  14(5.11) 20.7(10.4)19.3(6.47) 16.1(7.54) 10 hour 14.2(5.51)  20.1(11.93) 19.3(8.24) 19(8.17) 24 hour 14.5(4.69) 20.1(9.34) 16.7(6.09) 18.9(8.24)

TABLE 89 Pharmacokinetics Estrone End of Study (pg/mL) 4 μg 10 μg 25 μgPlacebo Post Dosing 4.328(2.7619) 4.643(2.5807) 6.652(11.508)4.363(2.5982)

Estrone Area Under the Curve (0-24 Hours)

TABLE 90 Estrone Area Under the Curve (0-24 hours) (h*pg/mL) 4 μg 10 μg25 μg Placebo Day 1 290.2(123.67) 462.7(195.64) 419.1(147.85)467.9(278.78) Day 14 326.6(114.09) 464.1(243.92) 428.7(161.75)426.8(180.67)

TABLE 91 Estrone Area Under the Curve (0-24 hours) (Baseline Adjusted)(h*pg/mL) 4 μg 10 μg 25 μg Placebo Day 1 7.2(20.91) 10.9(24.55)44.3(54.27) 43.5(97.41) Day 14  15(41.53) 43.2(84.87) 55.6(78.06)17.4(45.27)

TABLE 92 Estrone Area Under the Curve (0-24 hours) P-values PairwiseTest vs. 4 μg 10 μg 25 μg Day 1 0.003 0.0076 Day 14 0.042 0.0393

TABLE 93 Estrone Area Under the Curve (0-24 hours) P-values PairwiseTest vs. Placebo 4 μg 10 μg 25 μg Day 1 0.0193 0.9487 0.519  Day 140.0621 0.6117 0.9738

TABLE 94 Estrone Area Under the Curve (0-24 hours) P-values PairwiseTest vs. 4 μg (Baseline Adjusted) 10 μg 25 μg Day 1 0.6195 0.0104 Day 140.2251 0.0658

TABLE 95 Estrone Area Under the Curve (0-24 hours) P-values PairwiseTest vs. Placebo (Baseline Adjusted) 4 μg 10 μg 25 μg Day 1 0.13110.167  0.9761 Day 14 0.8721 0.2746 0.0886

TABLE 96 Estrone Area Under the Curve (0-24 hours) Ratio (Day 14) of Day14 to Day 1 4 μg 10 μg 25 μg Placebo AUC Ratio of Day 14 1.234 1.0231.039 1.006 to Day 1 (0.5824) (0.2675) (0.1941) (0.2316) Pairwise testvs — 0.1722 0.1866 — Pairwise test vs 0.1432 0.848  0.6544 — Placebo

Estrone C_(max)

TABLE 97 C_(max) (pg/mL) 4 μg 10 μg 25 μg Placebo Day 1 15.7(6.07)23.5(9.87)  21.9(7.73) 25.7(18.43) Day 14  16(5.5) 23.9(13.45)22.4(8.95) 22.8(10.89)

TABLE 98 C_(max) (Baseline Adjusted) (pg/mL) 4 μg 10 μg 25 μg PlaceboDay 1 0.4(3.05) 3.2(2.99) 5.1(4.78) 6.3(12.81) Day 14 0.6(3.49)3.7(8.79) 5.6(4.81) 3.4(5.69) 

TABLE 99 C_(max) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 1 0.0070.0126 Day 14 0.0301 0.0163

TABLE 100 C_(max) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μgDay 1 0.0373 0.6567 0.4223 Day 14 0.0275 0.7878 0.8979

TABLE 101 C_(max) P-values Pairwise Test vs. 4 μg (Baseline Adjusted) 10μg 25 μg Day 1 0.0087 0.0013 Day 14 0.1975 0.0014

TABLE 102 C_(max) P-values Pairwise Test vs. Placebo (Baseline Adjusted)4 μg 10 μg 25 μg Day 1 0.0659 0.3046 0.71 Day 14 0.0938 0.933 0.2249

TABLE 103 C_(max) Ratio (Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μgPlacebo C_(max) Ratio of Day 14 to 1.029 1.042 1.041 1.039 Day 1(0.2346) (0.3436) (0.2179) (0.2916) Pairwise test vs — 0.9035 0.8835 —Pairwise test vs 0.9188 0.9788 0.982 — Placebo

Estrone C_(avg)

TABLE 104 C_(avg) (pg/mL) 4 μg 10 μg 25 μg Placebo Day 1   13 (4.72)19.3 (8.15)  17.5 (6.16) 19.5 (11.62) Day 14 13.6 (4.75) 19.3 (10.16)17.9 (6.74) 17.8 (7.53) 

TABLE 105 C_(avg) (Baseline Adjusted) (pg/mL) 4 μg 10 μg 25 μg PlaceboDay 1 −2.3 (2.26) −1.1 (2.66) 0.7 (3.73) 0.1 (5.03) Day 14 −1.7 (3.25)−0.9 (5.91) 1.1 (4.81) −1.6 (3.8) 

TABLE 106 C_(avg) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.0075 0.0207 Day 14 0.042 0.0393

TABLE 107 C_(avg) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μgDay 1 0.0363 0.9487 0.519 Day 14 0.0621 0.6117 0.9738

TABLE 108 C_(avg) P-values Pairwise Test vs. 4 μg (Baseline Adjusted) 10μg 25 μg Day 1 0.1345 0.0057 Day 14 0.6351 0.0495

TABLE 109 C_(avg) P-values Pairwise Test vs. Placebo (Baseline Adjusted)4 μg 10 μg 25 μg Day 1 0.0712 0.3751 0.691 Day 14 0.912 0.7058 0.0742

TABLE 110 C_(avg) Ratio (Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μgPlacebo C_(avg) Ratio of Day 14 to 1.029 1.042 1.041 1.039 Day 1(0.2346) (0.3436) (0.2179) (0.2916) Pairwise test vs — 0.9035 0.8835 —Pairwise test vs 0.9188 0.9788 0.982 — Placebo

Estrone T_(max)

TABLE 111 T_(max) (h) 4 μg 10 μg 25 μg Placebo Day 1 14.1 (9.37) 11.9(9.76) 9.1 (7.43) 12.1 (9.39) Day 14 10.9 (9.03) 10.4 (8.93) 6.3 (6.9) 12.2 (9.24)

TABLE 112 T_(max) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.4862 0.0849 Day 14 0.8711 0.0982

TABLE 113 T_(max) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μgDay 1 0.5341 0.9449 0.2997 Day 14 0.6824 0.5639 0.0391

Estrone Conjugates

TABLE 114 Pharmacokinetics Estrone Conjugates Baseline (pg/mL) 4 μg 10μg 25 μg Placebo Baseline 250.3(162.91) 259.7(208.51) 374.4(586.45)280.7(171.26)

TABLE 115 Pharmacokinetics Estrone Conjugates Day 1 (pg/mL) 4 μg 10 μg25 μg Placebo Predose 225.1(215.01) 218.6(147.84) 312.4(410.38)271.2(153.33) 2 hour 206.8(163.2)  273.1(176.59) 396.6(408.16)223.4(162.11) 4 hour 241.7(176.87) 267.2(161.79) 413.3(343.25)241.8(139.77) 6 hour 240.6(181.14)  266(184.92) 477.8(472.66) 265(154.01) 10 hour   223(150.42) 243.5(173.71) 436.4(461)   258(133.21) 24 hour  229.4(186.79) 268.4(221.29) 306.4(322.91)268.8(153.22)

TABLE 116 Pharmacokinetics Estrone Conjugates Day 14 (pg/mL) 4 μg 10 μg25 μg Placebo Predose 212.7(140.19) 319.1(326.71) 411.1(624.14)256.1(133.07) 2 hour 212.4(145.02) 420.4(560.53) 434.3(491.31)285.6(158.61) 4 hour 240.2(155.7)  429.3(506.01) 505.1(618.47)273.1(148.76) 6 hour 225.8(164.76) 359.2(346.26) 483.8(515.95)267.7(181.53) 10 hour  238.3(152.45) 417.6(517.51) 492.5(598.16)306.9(178.68) 24 hour  206.4(154.26)  349(345.91) 309.6(380.88)240.1(115.84)

TABLE 117 Pharmacokinetics Estrone Conjugates End of Study (pg/mL) 4 μg10 μg 25 μg Placebo Post Dosing 237.4(151.19) 221.7(188.05)499.7(1089.67) 250(148.72)

Estrone Conjugates Area Under the Curve (0-24 Hours)

TABLE 118 Estrone Conjugates Area Under the Curve (0-24 hours) (h*pg/mL)4 μg 10 μg 25 μg Placebo Day 1 5077.5(3798.39) 5931.9(4209.95) 9126(9186.37) 5637.9(3151.49) Day 14 5172.9(3382.89)  8978(9811.23)9930.2(11711.99) 6275.2(3397.54)

TABLE 119 Estrone Conjugates Area Under the Curve (0-24 hours) (BaselineAdjusted) (h*pg/mL) 4 μg 10 μg 25 μg Placebo Day 1 375.5(843.98) 422.4(473.83) 2454.3(2600.25)  83(229.06) Day 14 660.5(1230.69)3767.2(7671.38)  3059(4792.46) 665.4(1552.19)

TABLE 120 Estrone Conjugates Area Under the Curve (0-24 hours) P-valuesPairwise Test vs. 4 μg 10 μg 25 μg Day 1 0.5219 0.0931 Day 14 0.13920.1166

TABLE 121 Estrone Conjugates Area Under the Curve (0-24 hours) P-valuesPairwise Test vs. Placebo 4 μg 10 μg 25 μg Day 1 0.639 0.8157 0.1472 Day14 0.3503 0.2898 0.2246

TABLE 122 Estrone Conjugates Area Under the Curve (0-24 hours) P-valuesPairwise Test vs. 4 μg (Baseline Adjusted) 10 μg 25 μg Day 1 0.83490.0028 Day 14 0.1087 0.0537

TABLE 123 Estrone Conjugates Area Under the Curve (0-24 hours) P- valuesPairwise Test vs. Placebo (Baseline Adjusted) 4 μg 10 μg 25 μg Day 10.1894 0.0134 0.001 Day 14 0.992 0.1225 0.0654

TABLE 124 Estrone Conjugates Area Under the Curve (0-24 hours) Ratio(Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μg Placebo AUC Ratio of1.115(0.4539) 1.444(1.0121) 1.107(0.3545) 1.125(0.4522) Day 14 to Day 1Pairwise test vs — 0.2279 0.9587 — Pairwise test vs 0.9459 0.2427 0.8975— Placebo

Estrone Conjugates C_(max)

TABLE 125 C_(max) (pg/mL) 4 μg 10 μg 25 μg Placebo Day 1 273.1(196.36)329.4(226.58) 542.1(475.49) 309.8(146.07) Day 14  289(183.79)511.7(568.75) 579.5(610.1)  343.6(182.2) 

TABLE 126 C_(max) (Baseline Adjusted) (pg/mL) 4 μg 10 μg 25 μg PlaceboDay 1 35.4(89.09)  90.2(65.2)  198.6(301.53) 27.1(49.69) Day 1448.2(132.61) 277.8(493.64) 236.1(372.42)   67(121.81)

TABLE 127 C_(max) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.4261 0.0333 Day 14 0.1332 0.0685

TABLE 128 C_(max) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μgDay 1 0.5369 0.7629 0.0625 Day 14 0.3902 0.2533 0.1356

TABLE 129 C_(max) P-values Pairwise Test vs. 4 μg (Baseline Adjusted) 10μg 25 μg Day 1 0.039 0.0345 Day 14 0.0726 0.0579

TABLE 130 C_(max) P-values Pairwise Test vs. Placebo (Baseline Adjusted)4 μg 10 μg 25 μg Day 1 0.7444 0.0033 0.0318 Day 14 0.6735 0.1065 0.0928

TABLE 131 C_(max) Ratio (Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μgPlacebo C_(max) Ratio of 1.13 1.524 1.144 (0.4569) 1.11 (0.5404) Day 14to (0.4068) (1.1682) Day 1 Pairwise test vs — 0.1969 0.9226 — Pairwisetest vs 0.9043 0.1919 0.8406 — Placebo

Estrone Conjugates C_(avg)

TABLE 132 C_(avg) (pg/mL) 4 μg 10 μg 25 μg Placebo Day 215.9 (154.77)247.2 (175.41) 380.3 (382.77) 244.6 (128.1) 1 Day 215.5 (140.95) 374.1(408.8) 413.8 (488) 261.5 (141.56) 14

TABLE 133 C_(avg) (Baseline Adjusted) (pg/mL) 4 μg 10 μg 25 μg PlaceboDay −21.8 (88.41)     8 (34.21) 36.8 (291.72) −33.7 (46.95) 1 Day −25.3(120.69) 140.2 (330.6) 70.3 (300.36)  −7.9 (89.89) 14

TABLE 134 C_(avg) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.5701 0.1004 Day 14 0.1392 0.1166

TABLE 135 C_(avg) P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μgDay 1 0.5562 0.9602 0.1741 Day 14 0.3503 0.2898 0.2246

TABLE 136 C_(avg) P-values Pairwise Test vs. 4 μg (Baseline Adjusted) 10μg 25 μg Day 1 0.1804 0.4201 Day 14 0.0606 0.2305

TABLE 137 C_(avg) P-values Pairwise Test vs. Placebo (Baseline Adjusted)4 μg 10 μg 25 μg Day 1 0.6353 0.0047 0.3473 Day 14 0.6439 0.0928 0.3244

TABLE 138 C_(avg) Ratio (Day 14) of Day 14 to Day 1 4 μg 10 μg 25 μgPlacebo C_(avg) Ratio of 1.13 (0.4068) 1.524 1.144 1.11 (0.5404) Day 14to (1.1682) (0.4569) Day 1 Pairwise test vs — 0.1969 0.9226 — Pairwisetest vs 0.9043 0.1919 0.8406 — Placebo

Estrone Conjugates T_(max)

TABLE 139 T_(max) (h) 4 μg 10 μg 25 μg Placebo Day 1 10.9 (8.66) 9.2(9.25) 5.4 (2.64) 13.1 (9.7) Day 14  8.4 (7.79)  9 (8.6) 5.9 (2.87)  8.1(6.76)

TABLE 140 T_(max) P-values Pairwise Test vs. 4 μg 10 μg 25 μg Day 10.5609 0.0154 Day 14 0.8173 0.2178

TABLE 141 Tmax P-values Pairwise Test vs. Placebo 4 μg 10 μg 25 μg Day 10.4893 0.2253 0.003 Day 14 0.9256 0.739 0.2087

In the phase 3 trial, all doses of TX-004HR compared with placebo (MITTn=747) significantly improved the 4 co-primary endpoints at week 2through week 12, as well as the secondary endpoints of vaginal drynessby week 6 and vulvar and/or vaginal itching or irritation by week 12(except 4 μg, p=0.0503), and was well-tolerated with notreatment-related serious AEs reported. The phase 3 PK study (n=72)showed no difference in systemic E2 levels for 4 μg and 10 μg TX-004HRvs placebo, as measured by AUC and C_(avg). E2 AUC and C_(avg) with 25μg TX-004HR was higher than placebo, but average concentrations remainedwithin the normal postmenopausal range (Table 142). E2 levels at day 84were similar to placebo indicating no systemic drug accumulation. SHBGconcentrations did not change with treatment. The two phase 2 studies(n=36 for each) of TX-004HR 10 μg and 25 μg resulted in statisticallysignificantly lower E2 absorption than an approved E2 tablet atidentical doses, with 25 μg TX-004HR demonstrating AUC less than ⅓ thatof the approved product (Table 143).

TABLE 142 Phase 3 study PK parameters for E2 (unadjusted mean ± SD).AUC₀₋₂₄ (pg * hr/mL) C_(avg) (pg/mL) Day Dose (μg) TX-004HR Placebop-value TX-004HR Placebo p-value 1 4  91.7 ± 37.9 116.6 ± 77.3 NS 3.92 ±1.46 4.86 ± 3.22 NS 10 138.2 ± 75.2 116.6 ± 77.3 NS 5.76 ± 3.13 4.86 ±3.22 NS 25 217.4 ± 99.0 116.6 ± 77.3 0.0021 9.06 ± 4.13 4.86 ± 3.220.0021 14 4  87.2 ± 42.8 104.2 ± 66.4 NS 3.63 ± 1.78 4.34 ± 2.77 NS 10110.1 ± 54.6 104.2 ± 66.4 NS 4.59 ± 2.27 4.34 ± 2.77 NS 25 171.6 ± 80.1104.2 ± 66.4 0.0108 7.15 ± 3.34 4.34 ± 2.77 0.0108

TABLE 143 Phase 2 studies PK parameters for E2 (baseline adjustedgeometric mean). AUC₀₋₂₄ (pg * hr/mL) C_(max) (pg/mL) Dose VaginalVaginal (μg) TX-004HR Tablet p-value TX-004HR Tablet p-value 10 49.62132.92 <0.0001 14.38 20.38 0.0194 25 89.21 292.06 <0.0001 23.08 42.70<0.0001

With robust efficacy demonstrated as early as 2 weeks and up to 12 weeksat all 3 doses, TX-004HR 4 μg and 10 μg showed negligible systemic E2absorption, while 25 μg resulted in very low systemic absorption of E2in the phase 3 trial. TX-004HR 10 μg and 25 μg showed lower systemic E2exposure than equivalent doses of an approved E2 tablet. The absence ofclinically meaningful increases in E2 concentrations paired with dataconsistent with a lack of systemic effects (e.g., no increase in SHBG)shows that TX-004 HR delivers excellent efficacy with negligible to verylow systemic exposure.

The impact of normal daily activities for 4 hours post dose wasevaluated, in comparison with the impact of remaining in the supineposition for 4 hours post dose on the pharmacokinetic (PK) profile ofTX-004HR 25 mcg. In two studies, at the same site, the same sixteenhealthy postmenopausal female subjects were fasted for at least 10 hoursprior to dosing through 4 hours following dosing. Subjects received a 25mcg dose of TX-004HR administered intravaginally by trained female studypersonnel. Following their first dose, the subjects were required toremain in a supine position for 4 hours following dosing. Following thesecond dose, after 5 minutes resting time, the subjects were instructedto be ambulatory in the clinic and refrain from reclining for the 4hours following dosing. Blood samples were collected at pre-definedintervals up to 24 hours after dosing. Plasma samples were analyzed forestradiol using LC-MS/MS. See, e.g., FIG. 23. PK parameters werecalculated on an individual and group mean basis with baselinecorrection.

The mean C_(max) and AUC₀₋₂₄ of estradiol was not significantlydifferent with ambulation than with supination. On an individual subjectbasis, the majority showed similar C_(max) and AUC₀₋₂₄ levels withambulation as with supination. There were no signs of posture having aneffect on absorption rate as evidenced by the similarity in groupaverage and individual subject T_(max). In addition, there was nodifference between the group mean profiles when compared on anindividual time point basis, further demonstrating that posture had noeffect on absorption. The systemic exposure of estradiol in TX-004HR 25mcg was generally low and occurred regardless of whether the subjectswere ambulatory or supine for 4 hours after dosing. An importantadvantage of the formulation is that a woman can be ambulatory almostimmediately after the formulation is administered, as opposed to otherknown formulations that require a subject to remain in a supine positionafter administration. Generally, other known formulations directadministration before bed at night because of the requirement to besupine, which requirement is unnecessary in the pharmaceuticalcompositions disclosed herein because the pharmaceutical compositionsdisclosed herein adhere to the vaginal tissue, the capsule dissolvesrapidly, and the formulation is released into the vagina and rapidlyabsorbed by the vaginal tissue. Because activity level does notadversely affect the systemic absorption of estradiol, the formulationof the invention gives the patient more flexibility with her dosingregimen.

Example 13 Safety Results in Randomized, Double-Blind,Placebo-Controlled Multicenter Study

Safety endpoints in the study included vital signs, clinical laboratorytests (blood chemistry, hematology, hormone levels, urine analysis),ECG, physical and gynecological examination findings, pap smears,endometrial biopsies, and adverse events (AEs). AEs included undesirablemedical conditions occurring at any time during all study phasesincluding the washout period, whether or not a study treatment had beenadministered. An AE was considered treatment emergent if it occurredafter study drug administration, or if it was pre-existing and worsenedduring 120 days post-dose follow up. Participants were given a diarywith instructions to record product use, sexual activity,symptoms/complaints, and other medications. AEs, concomitantmedications, and vital signs were recorded and assessed at each studyvisit from screening to week 12.

TX-004HR had a favorable safety profile and was well tolerated. Noclinically significant differences in AEs were observed betweentreatment and placebo groups (Table 144). Headache was the most commonlyreported TEAE, followed by vaginal discharge, nasopharyngitis, andvulvovaginal pruritus (Table 144). Headache was the onlytreatment-related TEAE that was numerically more frequent in womenreceiving TX-004HR than those receiving placebo (3.7% for 4-μg dose vs3.1% for placebo). Vaginal discharge was reported by numerically fewerwomen in any of the TX-004HR groups than by women in the placebo group.Most TEAEs were mild to moderate in severity. Few participants (1.8%)discontinued the study due to AEs.

TABLE 144 Number (%) of treatment emergent adverse events (TEAE)reported for ≥3% in any treatment arm of the safety population. TX- TX-TX-004HR 004HR 004HR 4 μg 10 μg 25 μg Placebo Preferred Term (n = 191)(n = 191) (n = 190) (n = 192) Any subject with 97 (50.8) 94 (49.2) 93(48.9) 111 (57.8) reported TEAE Headache 12 (6.3)  14 (7.3)  6 (3.2) 15(7.8) Vaginal discharge 5 (2.6) 6 (3.1) 4 (2.1) 13 (6.8) Nasopharyngitis5 (2.6) 6 (3.1) 7 (3.7) 10 (5.2) Vulvovaginal pruritus 4 (2.1) 3 (1.6) 7(3.7) 10 (5.2) Back pain 9 (4.7) 1 (0.5) 4 (2.1)  8 (4.2) Urinary tractinfection 5 (2.6) 5 (2.6) 8 (4.2)  4 (2.1) Upper respiratory tract 5(2.6) 6 (3.1) 3 (1.6)  5 (2.6) infection Oropharyngeal pain 1 (0.5) 0(0)   6 (3.2)  1 (0.5)

Nine serious TEAEs were reported in 8 subjects; however, none wereconsidered related to treatment. Complete heart block, appendicitis,endophthalmitis, and chronic obstructive pulmonary disease were eachreported by a different participant in the 25 μg group. Sinus nodedysfunction and ankle fracture were both reported for one women, andarthralgia and malignant melanoma were each reported for one women inthe 10 μg group. None of the women in the 4 μg group had reports ofserious TEAEs. One woman in the placebo group was reported to have acervical myelopathy. No deaths occurred during the study.

No diagnoses of endometrial hyperplasia or malignancy from endometrialbiopsies were observed at week 12. Total cholesterol numericallydecreased from baseline to week 12 by a mean of 0.024 mmol/L to 0.07mmol/L in the treatment groups, and by 0.008 mmol/L in the placebogroup. No clinically meaningful increases in triglycerides were observedin any active treatment groups compared with placebo. Sex hormonebinding globulin (SHBG) concentrations (measured in a subset of 72women) did not increase with treatment relative to placebo or baselineat week 12. No clinically significant changes in any laboratoryparameters were found.

The phase 3 clinical trial demonstrated that TX-004HR at 4 μg, 10 μg,and 25 μg doses is safe and effective for treating vaginal changes andself-reported symptoms of VVA in postmenopausal women. Statisticallysignificant and clinically meaningful improvements in all of the 4pre-specified co-primary endpoints (increase in the percentage ofvaginal superficial cells, decrease in the percentage of vaginalparabasal cells and vaginal pH, and decrease in severity of the MBS ofdyspareunia) occurred as early as 2 weeks with all 3 doses of TX-004HRas compared with placebo, and were sustained throughout the 12-weektrial. Additionally, improvements were found for the secondary endpointsof vaginal dryness and vulvar or vaginal irritation and itching. Theseimprovements were achieved without increasing systemic estrogenconcentrations (4 μg and 10 μg) or with negligible (25 μg) systemicestrogen exposure, as found in pharmacokinetic studies. TX-004HR wasalso well-tolerated with no clinically significant differences foundbetween treatment and placebo groups in any AEs or treatment-relatedAEs, and no treatment-related serious AEs.

The results demonstrate early onset of action in the clinical signs ofVVA with statistically significantly improved changes compared withplacebo. The efficacy results here were somewhat numerically higher thandata from a 12-week, randomized, controlled trial that compared a 10-μgvaginal estradiol tablet with placebo, which showed significantimprovements in the percentages of superficial and parabasal cells, andin pH compared with placebo (see, Simon et al. Obstet Gynecol. 2008;112:1053-1060). At 12 weeks, improvements were smaller with the 10-μgestradiol tablet (change of 13% in superficial cells, −37% in parabasalcells, and −1.3 in vaginal pH) than what was observed in this study withthe 10-μg TX-004HR dose (change of 17% in superficial cells, −44% inparabasal cells, and −1.4 in vaginal pH). While improvements in someobjective (cell and pH) endpoints were seen with the estradiol tabletwithin 2 weeks of treatment, the patient-reported improvements in acomposite score of subjective symptoms were not observed until 8 weeksof therapy, which can be perceived as a disadvantage for many users.That clinical trial did not assess individual symptoms. A secondrandomized, controlled trial of 10-μg and 25-μg estradiol tabletssimilarly did not find significant improvements over placebo in thecomposite score of vaginal symptoms with either dose until 7 weeks oftreatment (week 2, NS). Likewise, the SERM, ospemifene, was evaluated ina clinical trial for the treatment of dyspareunia, and statisticallysignificant improvements were not observed until week 12. See, Bachmannet al. Obstet Gynecol. 2008; 111:67-76; Portman et al. Menopause. 2013;20:623-630.

Importantly, the results reported here showed significant improvement indyspareunia within 2 weeks with all 3 doses of TX-004HR, with reductionsin severity scores from 1.5 to 1.7 points at week 12, which werecomparable or superior to reductions of 1.2 to 1.6 points reported forother currently approved dyspareunia treatments. See, VAGIFEM®(estradiol vaginal tablets) Prescribing Information. Bagsvaerd, Denmark:Novo Nordisk Pharmaceuticals Inc.; 2012; PREMARIN® (conjugated estrogenstablets, USP) Prescribing Information. Philadelphia, Pa.: WyethPharmaceuticals Inc.; 2010; OSPHENA® (ospemifene) tablets, for oral use.Prescribing Information. Shionogi, Inc. 2013.

Additionally, vaginal dryness improved from week 2 with 10 μg and 25 μgTX-004HR. None of the currently available products reported as early anonset of action for the symptom of vaginal dryness associated with VVAas did TX-004HR. Furthermore, TX-004HR 10 μg and 25 μg showedsignificant improvement in vaginal irritation and/or itching at week 12,while none of the currently available products on the market arereported to improve these symptoms. See, Portman et al. Maturitas. 2014;78:91-98; Eriksen et al. Eur J Obstet Gynecol Reprod Biol. 1992;44:137-144.

Based on a large survey of postmenopausal women in the United States,only a small proportion (7%) of women are thought to receiveprescription vaginal estrogen therapy alone for their VVA, probably dueto lack of information about available treatments, avoidance ofdiscussion of the topic with health care practitioners, ordissatisfaction with currently available products (see, e.g., Kingsberget al. J Sex Med. 2013; 10:1790-1799). Eliminating the need for anapplicator or individually measuring doses is intended to give women amore positive user experience and thus potentially better compliance,resulting in overall better efficacy of treatment.

The results with TX-004HR in this study exemplify one of the advantagesof local vaginal estrogen therapies: rapid symptom resolution withoutincreasing systemic estrogen concentrations. The mean area under theconcentration-time curve (AUC) and average concentration (C_(avg)) forestradiol were not significantly different from placebo with 4 μg and 10μg TX-004HR. Although statistically higher AUC for estradiol wasobserved with the 25 μg dose, estradiol levels remained within thepostmenopausal range with no evidence of accumulation by day 84.Although there was negligible systemic absorption, rapid efficacy wasobserved within 2 weeks of dosing with all doses of TX-004HR.

TX-004HR was well-tolerated. The 4 most commonly reported TEAEs,including vaginal discharge and vulvovaginal pruritus, were experiencedby fewer women in any TX-004HR group than in the placebo group, and weremostly mild to moderate in severity. By comparison, in a 12-week studyof the efficacy of ospemifene, vaginal discharge was reported more than6-times more frequently in the ospemifene group than in the placebogroup (see, Portman et al. Menopause. 2013; 20:623-630). Genitalpruritus was also reported 4-times more frequently in women treated withVagifem 10-μg tablets than with placebo in a 12-month randomized study(see, Vagifem® (estradiol vaginal tablets) Prescribing Information.Bagsvaerd, Denmark: Novo Nordisk Pharmaceuticals Inc.; 2012).Importantly, endometrial findings after TX-004HR were benign as nohyperplasia or malignancies were reported in biopsies at 12 weeks. Onsetof effect was seen as early as 2 weeks and was maintained throughout thestudy. TX-004HR was well tolerated as reported here and systemicestrogen exposure was negligible to very low as demonstrated by thepharmacokinetic study.

Example 14 Results of Female Sexual Function Index in Randomized,Double-Blind, Placebo-Controlled Multicenter Study

The trial was a randomized, double-blind, placebo controlled,multicenter, phase 3 study. Treatments were self-administered vaginally,once daily, for 2 weeks and then twice weekly, for 10 weeks. Femalesexual dysfunction (FSD) was evaluated using the multidimensional FemaleSexual Function Index (FSFI) at baseline and at week 12. The FSFI is abrief, validated, self-reporting questionnaire consisting of 19questions designed to assess the areas of arousal, desire, orgasm,lubrication, and pain. The Index defines sexual dysfunction by a totalFSFI score (the sum of the individual domain scores) of ≤26.55 out of apossible maximum score of 36.

Postmenopausal women (40-75 years ; BMI≤38 kg/m2) were included if theyhad ≤5% superficial cells on vaginal cytological smear; vaginal pH>5.0;self-identified most bothersome symptom (MBS) of moderate-to severedyspareunia; and anticipated sexual activity (with vaginal penetration)during the trial period. Vulvar and vaginal atrophy (VVA) treatments,including vaginal lubricants and moisturizers, were discontinued within7 days prior to screening. Use of oral estrogen-, progestin-, androgen-,or SERM-containing drug products were prohibited within 8 weeks of studystart. Changes from baseline in total and individual domain FSFI scoresfor each dose were compared with placebo using ANCOVA with baseline as acovariate.

764 postmenopausal women were randomized to 4 μg (n=191), 10 μg (n=191),or 25 μg (n=190) vaginal estradiol softgel capsules or placebo (n=192).The majority of the women were white (87%) with a mean age of 59 yearsand a mean BMI of 26.7 kg/m² (Table 145). The FSFI questionnaire wascompleted by those who were not in the PK sub-study (n=692; 90.6%). Theaverage baseline total FSFI score of 14.8 for all women indicated FSD inthe subjects.

TABLE 145 Summary of subjects enrolled in study Composition 4Composition 5 Composition 6 4 μg 10 μg 25 μg Composition 7 (n = 186) (n= 188) (n = 186) (n = 187) Age, years Mean ± SD 59.8 ± 6.0 58.6 ± 6.358.8 ± 6.2 59.4 ± 6.0 Race, n (%) White 162 (87.1) 165 (87.8)  161(86.6)  160 (85.6)  Black or African American 20 (10.8) 21 (11.2) 24(12.9) 21 (11.2) Asian 3 (1.6) 2 (1.1) 1 (0.5) 1 (0.5) BMI, kg/m² Mean ±SD 26.6 ± 4.9 26.8 ± 4.7 26.9 ± 4.8 26.6 ± 4.6 Baseline total FSFI ScoreMean ± SD  14.8 ± 6.13  15.8 ± 6.24  14.2 ± 6.21  14.4 ± 6.61 BaselineFSFI Pain Score Mean ± SD  1.6 ± 1.11  1.8 ± 1.22  1.7 ± 1.17  1.7 ±1.20

The Female Sexual Function Index (FSFI) total summary score is anumerically continuous measure that was descriptively summarized atVisits 2 and 6 and the change in the total summary score (Visit 6 minusVisit 2) was also descriptively summarized. The domain sub-scores andthe changes in the domain sub-scores were also descriptively summarized.Summaries were by treatment arm, and all active treatment arms combined.

In addition, the change in mean from baseline of each active treatmentgroup from the placebo group for each numerically continuous endpointwas evaluated. The least square (LS) mean changes and the 95% CI for thedifference in LS Mean changes between treated and placebo are provided.The FSFI Questionnaire consists of 19 questions divided among 6 domains,and has a minimum total score of 2.0 and a maximum score of 36.0 points.The FSFI questionnaire was administered to the randomized populationexcept for those subjects in the PK sub-study. At Baseline, the overallmean Total Score was 14.8 (14.8 for the 4 μg group; 15.8 for the 10 μggroup ; 14.2 for the 25 μg group; and 14.4 for the placebo group). TheLS mean change in the FSFI Total Score and domain scores from Baselineto Week 12 are summarized in Table 146.

Change from Baseline to Week 12 in FSFI total score and domains comparedto placebo was assessed.

After 12 weeks, total FSFI scores numerically improved from baseline inall groups, including placebo. Total FSFI score significantly increasedwith the 10 μg group (P<0.05) and the 25 μg group (P=0.0019) versusplacebo (FIG. 24).

FSFI lubrication and pain domain scores improved numerically in allgroups including placebo from baseline to 12 weeks; improvements for the10 μg group and the 25 μg group were statistically significantly greaterthan with placebo (FIG. 25A). The 25 μg composition significantlyimproved FSFI arousal (P=0.0085) and satisfaction (P=0.0073) domainscores at 12 weeks (FIG. 25B, FIG. 25C). All three doses were comparableto placebo in their effect on the FSFI domains of desire and orgasm(FIG. 25D, FIG. 25E). The 4 μg composition and placebo provided similarlevels of improvement. The compositions improved FSFI in adose-dependent manner, with the 25 μg dose having the greatestimprovement. All three doses were efficacious, and the numericimprovement in subjective symptoms was highest for subjects in the 10and 25 μg groups. The observed placebo response could be attributed tothe coconut oil (Miglyol) in the formulation for the placebo and theestradiol compositions, which may also contribute to the observedbenefits.

TABLE 146 Female Sexual Function Index Total and Domain Scores: 4 μg 10μg 25 μg Placebo Category Score Mean SD Mean SD Mean SD Mean SD TotalBaseline 14.8 6.13 15.8 6.24 14.2 6.21 14.4 6.61 Week 12 22.6 8.4 24.87.59 24.8 7.59 22 8.54 Change 7.98 7.551 8.85 7.361 10.49 8.176 7.748.41 LS Mean 7.909 0.9075 9.431 0.0492 10.283 0.0019 7.458 — ArousalBaseline 2.8 1.44 2.9 1.43 2.7 1.5 2.7 1.41 Week 12 3.6 1.61 4.1 1.474.1 1.39 3.6 1.52 Change 0.88 1.615 1.16 1.632 1.43 1.646 1.02 1.607 LSMean 0.876 0.9777 1.288 0.0581 1.393 0.008 0.927 — Desire Baseline 2.61.01 2.7 1.13 2.6 1.09 2.7 1.07 Week 12 3.3 1.11 3.5 1.13 3.5 1.06 3.31.21 Change 0.64 1.065 0.78 1.113 0.87 1.105 0.62 1.102 LS Mean 0.626 10.801 0.2753 0.849 0.1139 0.628 — Lubrication Baseline 2.1 1.25 2.3 1.252 1.19 2 1.29 Week 12 3.9 1.84 4.4 1.56 4.3 1.65 3.6 1.77 Change 1.841.782 2.12 1.612 2.36 1.744 1.64 1.871 LS Mean 1.835 0.4023 2.243 0.00122.3 0.0003 1.591 — Orgasm Baseline 2.7 1.74 2.9 1.74 2.4 1.68 2.4 1.73Week 12 3.8 1.89 4.1 1.75 4.1 1.66 3.7 1.97 Change 1.12 1.93 1.09 1.8211.68 1.857 1.31 1.86 LS Mean 1.162 0.9978 1.273 0.9424 1.59 0.0763 1.189— Satisfaction Baseline 2.9 1.37 3.2 1.43 2.9 1.37 2.9 1.49 Week 12 4.21.54 4.4 1.37 4.6 1.35 4.1 1.55 Change 1.31 1.512 1.24 1.534 1.64 1.6131.23 1.661 LS Mean 1.256 0.8798 1.382 0.3484 1.628 0.0063 1.165 —

While the pharmaceutical compositions and methods have been described interms of what are presently considered to be practical and preferredembodiments, it is to be understood that the disclosure need not belimited to the disclosed embodiments. It is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the claims, the scope of which should be accorded the broadestinterpretation so as to encompass all such modifications and similarembodiments. This disclosure includes any and all embodiments of thefollowing claims.

What is claimed is:
 1. A pharmaceutical composition comprising 4 μg ofestradiol, wherein intravaginal administration of the composition oncedaily for two weeks to a patient having VVA produces, in a plasma samplefrom the patient, (a) an unadjusted average concentration (C_(avg)) ofestradiol ranging from 1.82 pg/mL to 5.38 pg/mL; and/or (b) anunadjusted peak plasma concentration (C_(max)) of estradiol ranging from2.49 pg/mL to 7.11 pg/mL.
 2. The pharmaceutical composition of claim 1,wherein intravaginal administration of the composition once daily fortwo weeks to a patient having VVA produces, in a plasma sample from thepatient, (a) an unadjusted C_(avg) of estradiol of about 3.6 pg/mL;and/or (b) an unadjusted C_(max) of estradiol of about 4.8 pg/mL.
 3. Thepharmaceutical composition of claim 1, wherein intravaginaladministration of the composition once daily for two weeks to a patienthaving VVA further produces, in a plasma sample from the patient, (c) anunadjusted area under the curve (AUC)₀₋₂₄ of estradiol ranging from44.43 pg*hr/mL to 129.97 pg*hr/mL.
 4. The pharmaceutical composition ofclaim 3, wherein intravaginal administration of the composition oncedaily for two weeks to a patient having VVA produces, in a plasma samplefrom the patient, (c) an unadjusted AUC₀₋₂₄ of estradiol of about 87.2pg*hr/mL.
 5. The pharmaceutical composition of claim 1, whereinestradiol is the only active hormone in the composition.
 6. A method oftreating moderate to severe dyspareunia, comprising intravaginallyadministering a pharmaceutical composition comprising 4 μg of estradiolto a patient having VVA once daily for two weeks, wherein administrationof the composition once daily for two weeks to the patient produces, ina plasma sample from the patient, (a) an unadjusted C_(avg) of estradiolranging from 1.82 pg/mL to 5.38 pg/mL; and/or (b) an unadjusted C_(max)of estradiol ranging from 2.49 pg/mL to 7.11 pg/mL.
 7. The method ofclaim 6, wherein administration of the composition once daily for twoweeks to a patient having VVA produces, in a plasma sample from thepatient, (a) an unadjusted C_(avg) of estradiol of about 3.6 pg/mL;and/or (b) an unadjusted C_(max) of estradiol of about 4.8 pg/mL.
 8. Themethod of claim 6, wherein administration of the composition once dailyfor two weeks to a patient having VVA further produces, in a plasmasample from the patient, (c) an unadjusted AUC₀₋₂₄ of estradiol rangingfrom 44.43 pg*hr/mL to 129.97 pg*hr/mL.
 9. The method of claim 8,wherein administration of the composition once daily for two weeks to apatient having VVA produces, in a plasma sample from the patient, (c) anunadjusted AUC₀₋₂₄ of estradiol of about 87.2 pg*hr/mL.
 10. Apharmaceutical composition comprising 10 μg of estradiol, whereinintravaginal administration of the composition once daily for two weeksto a patient having VVA produces, in a plasma sample from the patient,(a) an unadjusted C_(avg) of estradiol ranging from 2.33 pg/mL to 6.87pg/mL; and/or (b) an unadjusted C_(max) of estradiol ranging from 4.94pg/mL to 9.66 pg/mL.
 11. The pharmaceutical composition of claim 10,wherein intravaginal administration of the composition once daily fortwo weeks to a patient having VVA produces, in a plasma sample from thepatient, (a) an unadjusted C_(avg) of estradiol of about 4.6 pg/mL;and/or (b) an unadjusted C_(max) of estradiol of about 7.3 pg/mL. 12.The pharmaceutical composition of claim 10, wherein intravaginaladministration of the composition once daily for two weeks to a patienthaving VVA further produces, in a plasma sample from the patient, (c) anunadjusted AUC₀₋₂₄ of estradiol ranging from 55.53 pg*hr/mL to 164.67pg*hr/mL.
 13. The pharmaceutical composition of claim 12, whereinintravaginal administration of the composition once daily for two weeksto a patient having VVA produces, in a plasma sample from the patient,(c) an unadjusted AUC₀₋₂₄ of estradiol of about 110.1 pg*hr/mL.
 14. Thepharmaceutical composition of claim 10, wherein estradiol is the onlyactive hormone in the composition.
 15. A method of treating moderate tosevere dyspareunia, comprising intravaginally administering apharmaceutical composition comprising 10 μg of estradiol to a patienthaving VVA once daily for two weeks, wherein administration of thecomposition once daily for two weeks to the patient produces, in aplasma sample from the patient, (a) an unadjusted C_(avg) of estradiolranging from 2.33 pg/mL to 6.87 pg/mL; and/or (b) an unadjusted C_(max)of estradiol ranging from 4.94 pg/mL to 9.66 pg/mL.
 16. The method ofclaim 15, wherein administration of the composition once daily for twoweeks to a patient having VVA produces, in a plasma sample from thepatient, (a) an unadjusted C_(avg) of estradiol of about 4.6 pg/mL;and/or (b) an unadjusted C_(max) of estradiol of about 7.3 pg/mL. 17.The method of claim 15, wherein administration of the composition oncedaily for two weeks to a patient having VVA further produces, in aplasma sample from the patient, (c) an unadjusted AUC₀₋₂₄ of estradiolranging from 55.53 pg*hr/mL to 164.67 pg*hr/mL.
 18. The method of claim17, wherein administration of the composition once daily for two weeksto a patient having VVA produces, in a plasma sample from the patient,(c) an unadjusted AUC₀₋₂₄ of estradiol of about 110.1 pg*hr/mL.
 19. Apharmaceutical composition comprising 25 μg of estradiol, whereinintravaginal administration of the composition once daily for two weeksto a patient having VVA produces, in a plasma sample from the patient,(a) an unadjusted C_(avg) of estradiol ranging from 3.76 pg/mL to 10.44pg/mL; and/or (b) an unadjusted C_(max) of estradiol ranging from 8.09pg/mL to 23.31 pg/mL.
 20. The pharmaceutical composition of claim 19,wherein intravaginal administration of the composition once daily fortwo weeks to a patient having VVA produces, in a plasma sample from thepatient, (a) an unadjusted C_(avg) of estradiol of about 7.1 pg/mL;and/or (b) an unadjusted C_(max) of estradiol of about 15.7 pg/mL. 21.The pharmaceutical composition of claim 19, wherein intravaginaladministration of the composition once daily for two weeks to a patienthaving VVA further produces, in a plasma sample from the patient, (c) anunadjusted AUC-24 of estradiol ranging from 91.47 pg*hr/mL to 251.73pg*hr/mL.
 22. The pharmaceutical composition of claim 21, whereinintravaginal administration of the composition once daily for two weeksto a patient having VVA produces, in a plasma sample from the patient,(c) an unadjusted AUC-24 of estradiol of about 171.6 pg*hr/mL.
 23. Thepharmaceutical composition of claim 19, wherein estradiol is the onlyactive hormone in the composition.
 24. A method of treating moderate tosevere dyspareunia, comprising intravaginally administering apharmaceutical composition comprising 25 μg of estradiol to a patienthaving VVA once daily for two weeks, wherein administration of thecomposition once daily for two weeks to the patient produces, in aplasma sample from the patient, (a) an unadjusted C_(avg) of estradiolranging from 3.76 pg/mL to 10.44 pg/mL; and/or (b) an unadjusted C_(max)of estradiol ranging from 8.09 pg/mL 23.31 pg/mL.
 25. The method ofclaim 24, wherein administration of the composition once daily for twoweeks to a patient having VVA produces, in a plasma sample from thepatient, (a) an unadjusted C_(avg) of estradiol of about 7.1 pg/mL;and/or (b) an unadjusted C_(max) of estradiol of about 15.7 pg/mL. 26.The method of claim 24, wherein administration of the composition oncedaily for two weeks to a patient having VVA further produces, in aplasma sample from the patient, (c) an unadjusted AUC₀₋₂₄ of estradiolranging from 91.47 pg*hr/mL to 251.73 pg*hr/mL.
 27. The method of claim26, wherein administration of the composition once daily for two weeksto a patient having VVA produces, in a plasma sample from the patient,(c) an unadjusted AUC₀₋₂₄ of estradiol of about 171.6 pg*hr/mL.